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PHARMACEUTICO - ANALYTICAL AND TOXICOLOGICAL STUDY OF SHUDDHA MALLA ON WISTAR ALBINO RATS (AN EXPERIMENTAL STUDY), BY ...

PHARMACEUTICO - ANALYTICAL AND TOXICOLOGICAL STUDY OF SHUDDHA MALLA ON WISTAR ALBINO RATS (AN EXPERIMENTAL STUDY), BY
DR. K. PALLAVI, DEPARTMENT OF POST GRADUATE STUDIES IN RASASHASTRA, TARANATH GOVT. AYURVEDIC MEDICAL COLLEGE, BELLARY – 583 101

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    • PHARMACEUTICO - ANALYTICAL ANDTOXICOLOGICAL STUDY OF SHUDDHA MALLA ON WISTAR ALBINO RATS (AN EXPERIMENTAL STUDY) BY DR. K. PALLAVI Dissertation submitted to the RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE. In partial fulfillment of the requirements for the degree of AYURVEDA VACHASPATI (DOCTOR OF MEDICINE) In RASASHASTRA Under The Guidance of Dr.SHANKAR GOWDA M.D.(AYU) Asst.Professor T.G.A.M.C., Bellary . DEPARTMENT OF POST GRADUATE STUDIES IN RASASHASTRATARANATH GOVT. AYURVEDIC MEDICAL COLLEGE, BELLARY – 583 101 (KARNATAKA) 2008
    • RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE. DECLARATION BY THE CANDIDATEI hereby declare that this dissertation entitled :“Pharmaceutico – Analytical and toxicological Study ofShuddha Malla on Wistar albino rats (An ExperimentalStudy)” is a bonafide and genuine research work carried out by meunder the guidance of Dr.Shankara Gowda, M.D. (Ayu),Asst.Professor, T.G.A.M.C, Bellary. Signature of the CandidateDate : DR. K.PALLAVIPlace : Post Graduate Scholar in Rasashastra T.G.A.M.C, Bellary. 2
    • RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA , BANGALORE. CERTIFICATE BY THE GUIDE This is to certify that the dissertation entitled:“Pharmaceutico - Analytical and toxicological Study ofShuddha Malla on Wistar albino Rats -An ExperimentalStudy” is a bonafide research work done by DR.K.PALLAVI inpartial fulfillment of the requirement for the degree of AyurvedaVachaspati, Doctor of Medicine (Ayurveda). Signature of the GuideDate : Dr.SHANKARA GOWDA, M.D. (Ayu)Place : Asst. Professor T.G.A.M.C., Bellary. 3
    • RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE. ENDORSEMENT BY THE HOD, PRINCIPAL/ HEAD OF THE INSTITUTION This is to certify that the dissertation entitled“Pharmaceutico analytical and toxicological Study of ShuddhaMalla on Wistar Albino Rats-An Experimental Study” is abonafide research work done by DR.K.PALLAVI under theguidance of Dr.Shankara Gowda, M.D. (Ayu) Professor, T.G.A.M.C.Bellary.Seal & Signature of the H.O.D Seal & Signature of the PrincipalDr. M.S.DODDAMANI, M.D. (Ayu) Dr. K.VISHWAMBHARAM.D.(Ayu)Date : Date :Place : Place : 4
    • RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE. DECLARATION BY THE CANDIDATE I hereby declare that the Rajiv Gandhi University of HealthScience, Karnataka shall have the rights to preserve use anddisseminate this dissertation in print or electronic format foracademic / research purpose. Signature of the candidateDate : DR.K.PALLAVIPlace: 5
    • ACKNOWLEDGEMENT I pray and bow before almighty for having given me this unique opportunity. It gives me pleasure to express my gratitude with profound respect to my guideDr.Shankara Gowda, Asst.Professor, Dept of PG Studies in Rasashastra, T.G.A.M.C.,Bellary for his scholarly guidance, constant encouragement throughout my study. I am overwhelmingly grateful to Dr.M.S.Doddamani, Professor& HOD, Dept ofPost Graduate Studies in Rasashastra, T.G.A.M.C., Bellary for his moral encouragement,inspiration and continuous support in completing my work. I am blessed to have precious supervision, pinpoint suggestion and constant helpof Dr.N.B.Shridhar,MVSc,Phd Asst.Professor,Principal Investigator, Department ofPharmacology & toxicology, KVAFSU, who guided me during experimental study. I extend my gratefulness to honourable Principal Prof. K.Viswambhara M.D (Ayu)for providing all facilities to make the study success. I express my profound sense of gratitude to all the PG Scientific committeemembers Dr. Shobha Hiremath,Dr.Surekha Madikeri & Dr.Ravi Chavan for theircontinuous help & suggestions. I am extremely grateful to the respected & esteemed ,Dr.Sathyanarayana Bhat,Dr.G.R.Vastrad, Dr.L.N. Kundargi, Dr. V.L. Yadhalli, Dr. Hugar, Dr. Saraswathi,Dr.Shashikala Biradar, Dr. Rajshekar Ganiger, Dr. Srivatsa,Dr.Madhava Diggavi , fortheir inspiration and encouragement. I am cordially thankful to Prof.Subodh ,Prof.Subramanian ,Prof.Deshpande,Mr.Krishnamurthy, Dept.of Materials Engineering, Indian Institute of Science,Bangaloreand Ganesh Consultancy & Analytical Services for their kind co-operation in performinganalytical study. My Special thanks to Prof.Narayan Swamy, Dept of Pathology, KVAFSU for hiskind co-operation in histopathological study. I am thankful to Mr.Srinivas, Vijay Diagnostics centre for on time reports inhaematology & histopathology. 6
    • I am thankful to Dr .K.P.Suresh, Scientist (Biostatistics), National Institute ofAnimal Nutrition & Physiology, Bangalore, for his kind co-operation in procuringstatistical reports. Thanks to Mr. D.Vaman Rao, Professor in chemistry & metallurgy for his prudentdiscussions on drug analytical results. My thanks to Central Animal house, Indian Institute Of Science, Bangalore forproviding the experimental animals as needed.I convey my thanks to Dr.Shivanand, Dr.Shreelatha,Dr.MohanKumar,Vanitha,Vinod,KVAFSU for their kind assistance in performing experimental study at the locale. I wish to express my thanks to: Dr.Veerendra Hatti ,Dr.Manjula, Dr.Anuroopa,Dr.Nischitha,Dr.RamacharyaGudi,Dr.AjithNarayan,Dr.Usharani,Dr.Mamatha,Dr.Mallamma,Dr.SriMukunda,Dr.C.M.Joshi,Dr.AbdulKareem,Dr.Lajana,Dr.Sunitha,Dr.Brahmanand,Dr.Rohith,Dr.Srikanth,Dr.ShriRaj,Dr.Manjunath,Dr.PoornimaBhat,Dr.Prajnami,Dr.SunithaG.S,Dr.SunithaM.L,Dr.Shwetha,Dr.AjayKumar,Dr.Kishor,Dr.Shrinidhi & Dr.Manjunath for their co-operation during the study period. My special thanks to my batch mates, friends Dr.Revati.Huddar, Dr.SaritaraniDr.Sanjeeva gowda Patil, Dr.Naveen.K , Dr.Sandeep.S and Dr.ManjulaC.V for theiradorable assistance through out the study period. I wish to convey sincere gratitude to my beloved teachers Dr.Ashalatha,Dr.Ganesh Kumar & Dr.Vijayalakshmi who fed me with blessings. I am endeared to express my special thanks to Dr.K.V.Guruprasad who stood asa constant source of inspiration and assisted me throughout the study. Deep from my heart my gratitude to my lovable parents Smt & Shri G.RajivShetty who made my dream come true. I wish to thank my brothers and all my family members for their support inaccomplishing this work. My sincere thanks to CCRAS for having selected present thesis for grants. I am sincerely thankful to all teaching staffs, physicians, staff nurses and non-teaching staffs of T.G.A.M.C. Hospital, Bellary & all those who have helped me directlyor indirectly in completion of this dissertation & for their generous and kind help formaking this work a success. DR.K.PALLAVI 7
    • LIST OF ABBREVIATIONSA.P Ayurveda PrakashaA.N Adarsha NighantuAs ArsenicATO,As2O3 Arsenic tri oxideB.P Bhava PrakashaB.P.N Bhava Prakasha NighantuB.R.R.S Bruhad Rasaraja Sundarad 50 Diffraction 50% for particle Size.JCPDS Joint Committee For Powder Diffraction Standards.K.N Kaiyadeva NighantuLD 50 Lethal Dose 50/median lethal dose.M.N Madanapala NighantuMTD Maximum tolerated dose.NOAEL No observed adverse effect level.P PhosphorousR RasamritamR.C Rasendra ChudamaniR.N Raja NighantuR.T Rasa TaranginiR.R.S Rasa Ratna SamucchayaR.J.N Rasa Jala NidhiSPF Specific pathogen FreeY.R Yoga RatnakaraX-RD X- Ray Diffraction.XPDF X-ray powder diffraction files 8
    • TABLE OF CONTENTSSl No. CONTENTS PAGE No.I INTRODUCTION.II Aims & Objectives.III Review of Literature. • Malla-A classical Review. • Arsenic & Arsenic trioxide • Karavellaka & its Modern Review • Pharmaceutical Review. • Analytical Review. • Toxicology Review. • Experimental Review.IV Methodology. Pharmaceutical study. • Malla Shodhana In Dola Yantra Swedana. Analytical study. • Physico-chemical, AAS, XRD, Particle Size Experimental study. • Acute Study • Sub-Acute StudyV Results. • Acute Study • Sub-acute StudyVI Discussions. • Acute Study • Sub-acute StudyVII Conclusion.VIII Summary.IX Limitations & Scope for the study.X Bibliographic References.XI Annexure. 9
    • LIST OF TABLESSL. PAGE TABLESNO. NO.1. Synonyms of Malla according to various authors.2. Varieties of Malla based on colour.3. Varieties of Malla based on Appearance.4. Malla shodhana according to various authors.5. Gunas of Malla according to various authors.6. Synonyms of Karavellaka according to various authors.7. Pharmacological Properties of Karavellaka.8. Observations during Malla shodhana in Karavellaka Swarasa.9. Organoleptic Characters of two samples of Malla10. Summary of Particle Size analysis.11. Shows Results of X-RD studies.12. Shows Drugs according to groups in acute study.13. Shows Calculation of administered dose of each group.14. Shows Calculation of escalated doses for two groups.15. Shows the day and number of rats dead in group IV.16. Shows the day and number of rats dead in group VII.17. Shows drugs according to groups in Sub-acute study.18. Shows weight and calculation of doses.19. Shows the physico-chemical results of Samples20. Shows Arsenic estimation in the given samples.21. Shows comparison of body weight between groups.22. Shows the inter group differences and its significance.23. Shows the percentage of rats dead in group IV.24. Shows the percentage of rats dead in group VII.25. Shows the comparison of Hb between groups.26. Shows the comparison of TLC between groups.27. Shows the comparison of DC-P between groups.28. Shows the comparison of lymphocytes between groups.29. Shows the comparison of eosinophils between groups.30. Shows the comparison of monocytes between groups.31. Shows the comparison of PCV between the groups. 10
    • 32. Shows the comparison of RBC between groups.33. Shows the comparison of Urea between groups.34. Shows the comparison of Creatinine between groups.35. Shows the comparison of SGOT between the groups.36. Shows the comparison SGPT between the groups.37. Shows the histopathology results.38. Shows the body weight between groups.39. Shows the comparison of Hb between groups.40. Shows the comparison of total Count between groups.41. Shows the comparison of DC-P between groups.42. Shows the comparison of DC-L between groups.43. Shows the comparison of DC-E between groups.44. Shows the comparison of DC-M between the groups.45. Shows the comparison of RBC between the groups.46. Shows the comparison of PCV between the groups.47. Shows the comparison of SGOT between the groups.48. Shows the comparison of SGPT between the groups.49. Shows the comparison of albumin between the groups.50. Shows the comparison of TPR between the groups.51. Shows the comparison of creatinine between the groups.52. Shows the comparison of BUN between the groups.53. Shows the histopathological results of different groups. 11
    • LIST OF GRAPHSSL. GRAPHS PAGE NONO.1. XRD of Raw /Ashuddha Malla2. XRD of Shuddha Malla.3. Comparitive XRD peaks between Shuddha and Ashuddha Malla.4. Diffraction pattern of Particle Size of Shuddha Malla.5. Diffraction pattern of Particle Size of Ashuddha Malla.6. Shows the body weight difference in between the groups in acute.7. Representation of Quantal dose response in group IV.8. Representation of Quantal dose response in group VII9. Representation of body weight in between groups Subacute. 12
    • LIST OF ILLUSTRATIONS, FIGURES AND PHOTOGRAPHSSL.NO FIGURES OR PHOTOGRAPHS PAGE NO1. Arsenic inhibiting enzymes.2. Chemical Structure of ATO.3. Raw Malla.4. Karavellaka.5. Ground Karavellaka kalka.6. Extraction of Karavellaka swarasa.7. Measured karavellaka swarasa.8. Malla in pottali.9. Dola Yantra Swedana.10. Boiling swarasa after 2 hours.11. Boiling swarasa after 5 hours.12. Pottali opened after cooling.13. Washing Malla pieces in hot water.14. Powdering Malla.15. Rats in cage.16. Cages arranged.17. Feeding and water for rats.18. Dosage prepared.19. Weighing the animals.20. Analytical balance.21. Numbering of rats.22. Tuberculin syringe.23. Gavaging the dose.24. Ether chamber.25. Storage Vials.26. Preparation of ether chamber.27. Blood drawing.28. Capillary tube and blood stored.29. Centrifuging blood.30. Semi-auto analyzer.31. Dissection set.32. Diethyl ether used for anaesthesia.33. Micropipettes with serum.34. Clear Serum in a pipette.35. Reagent mixing.36. Rehabilitating rat after anaesthesia.37. Blood clot after centrifuge38. Separating Serum39. Rat in ether chamber.40. Anaesthetized Rat.41. Decapitation of rat.42. Dead rat .43. Exposing Fascia 13
    • 44. Excising the organs45. Dissection of Brain.46. Fixing organs in NBF47. Ready for Histopathology ACUTE STUDY-histopathology.48. Photo micrographs of Heart tissues.49. Photomicrographs of Brain.50. Photomicrographs of Liver.51. Photomicrographs of Kidney.52. Photomicrographs of Lungs.53. Photomicrographs of Spleen.54. Photomicrographs of Gastric mucosa. SUB-ACUTE STUDY-histopathology.55. Photo micrographs of Heart tissues.56. Photomicrographs of Brain.57. Photomicrographs of Liver.58. Photomicrographs of Kidney.59. Photomicrographs of Lungs.60. Photomicrographs of Spleen.61. Photomicrographs of Gastric mucosa.62. Collision in fragmented Particles.63. Effect of temperature & reaction.64. Effect of Concentration on collision. 14
    • ABSTRACTTitle: “Pharmaceutico - Analytical and toxicological study of Shuddha Malla On Wistar Albino Rats (An experimental Study).”Background: Malla is one of the Sadharana Rasa. Shodhana is not only purification but alsopharmaceutical, pharmacological enhancement and detoxification therapy..The drug wasthen used to screen the toxicity on Wistar albino rats through Acute and Sub-acute Study.Objectives: 1) Shodhana of Malla by swedana method. 2) Physico – chemical Analysis of Shuddha Malla. 3) To screen the probable toxicity of Shuddha Malla in repeated doses. 4) To screen the probable toxicity Shuddha Malla in a single dose. 5) To find the median lethal dose of Shuddha Malla & Ashuddha Malla.Methods:Pharmaceutical Study: Swedana method for 6 hrs in Karavellaka Swarasa.From this study 47 gm (94%) ofShuddha Malla was obtained from 50 gm of Raw Malla.Analytical study: Physical tests like Ash value etc., Loss on drying, pH, estimation of arsenic,Particle Size, X-RD studies were conducted on Ashuddha and Shuddha Malla. Estimation of As% was in significant Purity levels proving to be genuine. An X-RD study has shown composition of Malla as As2O3 in both the samples with differentstandards. Particle size varies which shows an increased size after shodhanaExperimental study: Toxicity studies both acute and Sub acute was carried out for 14 and 28days respectively. The Control with the placebo gum acacia was common in both thestudy.. The repeated doses of Shuddha and Ashuddha Malla also were administered toknow the significant changes in 2 different respective groups. The escalated doses of boththe drugs were carried out in order to find the LD50 during the acute study. Collection ofblood and necropsy of rats was carried out to extract the data analysis.Results: The 10 and 20 times the dose of therapeutic dose of Shuddha Malla proved to benon-toxic. The repeated daily therapeutic dose of Shuddha Malla also proved to be non-toxic. 15
    • Conclusion: ◊ Shuddha Malla reveals more of Arsenic %. ◊ The median lethal dose of Shuddha Malla is 16.65mg. ◊ The single dose, repeated therapeutic dose for 14 days and 28 days of Shuddha Malla is non-toxic.Key words: Shodhana, Chemical Analysis, X-RD, Toxicity, escalated dose, median lethal dose. 16
    • INTRODUCTION Rasashastra dealing with drug pharmacology holds a valid response of drugdesigning, formulating, analyzing with a vision to cure diseases. Never medicines have tobe invented, evolved. Probably the result was evolution of mineral medicines after 100sof years of hard work, observations, experiments and research. An article JAMA on 15th December 2004, made feel the necessity of safety dataon metallomineral and herbo-mineral formulations used in Ayurveda. Conventionally,modern toxicology and pharmacology consider all the heavy metals as highly toxic. Butthis toxicity the vishathva was tamed by our seers by means of Shodhana. On the contrarythey become potent therapeutically. Malla is one such wonder Rasadravya used in day to day practice included underSadharana Rasa contains Arsenic. On review it was found that pharmaceutical andanalytical studies are carried but not toxicological studies. Hence a positive hypothesis(H1) is made that, with classical therapeutic dosage the safety of Malla is to be knownand the need of the hour is to prove its safety use through toxicological studies. So the present work entitled “PHARMACEUTICO – ANALYTICAL ANDTOXICOLOGICAL STUDY OF SHUDDHA MALLA ON WISTAR ALBINORATS(AN EXPERIMENTAL STUDY)’’is taken up in the interest of producingexperimental base for safety clinical use of Shuddha Malla .The present study has been categorized into 1. Introduction: Explains need of the study and hypothesis behind the study. 2. Objectives: Substantiating the mode of study 3. Review of literature: Reveals the drug review, pharmaceutical review, analytical review Experimental review and toxicological review. 4. Methodology: Is divided into three parts Pharmaceutical study: 17
    • Includes detailed documentation of Shodhana of Malla by Karavellaka swarasa carried out. Analytical study: Includes physico – chemical Analysis, estimation of Arsenic in the Sample along with particle Size and X-Ray Diffraction Study. Conducted on Ashodhita Malla and Karavellaka Shodhita Malla. Experimental study: Includes screening of probable toxicity of Shuddha Malla and Ashuddha Malla on Wistar albino Rats. It is in two sections as Acute and Sub-acute Study.5. Results: Includes results of Pharmaceutical, Analytical study and Statistical reports of Experimental Toxicological Studies both acute and Sub-acute.6. Discussion: Includes observations, findings & results of study along with probable Explanations and theories.7. Conclusion: A view of concluding points.8. Summary: The whole study is summarized.9. Bibliographical references: List of references from classical, modern texts and websites. 18
    • OBJECTIVES1) To carry out Shodhana of Malla in Karavellaka Swarasa.2) To carry out Physico-chemical analysis of Shuddha Malla.3) To carry out Acute toxicity Study of Shuddha Malla and Ashuddha Malla on Wistar albino rats.4) To carry out Sub-acute toxicity Study of Shuddha Malla and Ashuddha Malla Wistar albino rats.5) To carry out dose-dependent study and evaluate LD 50 of both Shuddha Malla and Ashuddha Malla.6) To carry out the relevant Serum and haematological laboratory investigations.7) To carry out histopathological analysis to understand organ toxicity.8) To prove the Safety dose of the drug. 19
    • MALLA Malla is one of essential Sadharana Rasas1. Most of Rasagranthas considered itunder Sadharana Rasa. Malla as a drug molecule was found in Samhitha kala itself. Itwas named as Phenashma and explained along with Haratala and both of them areconsidered as Dhatuvisha2.Nirukti: 3, 4 “Mallati dharati balamiti” The one which does baladharana, Is strong, robust, to hold, Possess, Excellent.Nishpatti: 5 The word Malla is derived from ‘Mall’ Dhatu and ‘Ach’ Pratyaya.Varga6: It is placed under Sadharana Rasa Varga.Historical Review: • Mentioned first as Phenashma along with Haratala as Dhatuvisha2. • Most of Rasacharyas explained it under Sadharana Rasavarga.Occurrence7: It is scarcely available along with the ores of Iron, Copper, and Sulphuretc. It is also available as a component in Haratala and Manahshila.If occurs in mineralform it has traces of antimony, nickel, silver, iron, bismuth and gold.Availability8: In the mineral form in China, Europe etc.In India Hazaaribagh (Bihar),Chitral (Kashmir) and nearby areas obtained with ores of Makshika etc.The artificialforms are from chimneys of industries and the places where Arsenopyrites are burnt.Characteristics9: Malla is crystalline or amorphous substance, white in colour. Itspowder resembles the flour of wheat but is much heavier. The surface of Malla has apeculiar shine and sometimes there occurs a yellowish tinge.Physical Properties9: When heated, Malla becomes soft and emits the odour like garlic.One crystalline variety of Malla is soluble in water, in very small quantity. The crystalsof Arsenic oxide are Octahedral or Monoclinic, of which the latter is unstable. Whenheated with intense heat, it evaporates in the atmosphere, directly (sublimation) givingout garlic odour.Grahya Malla10: Sphatikabha Malla is of best quality. Shankhabha or Shwethabha Malla is of better quality. Haridrabha Malla is of Good quality. 20
    • Vernacular names11:Kannada : Shankha pashana. Hindi : Shankhia.English : White Arsenic. Latin : Arsenicum Album.Gujarati : Somala, Sankhiyo. Marathi : Somalakhara.Bengali : Sankhavisa. Arabian : Sanmuluphar.Synonyms: Various synonyms have been given for Malla in Rasashastra classics basedon its colour, appearance, occurrence and action. Ex: Phenashma means white stone2.Sphatikabha, it looks like Sphatika mani12.Table No. 1: Shows the Synonyms of Malla.SYNONYMS R.J.N13 B.R.R.S14 R.R.S15 R.T16 Y.R17Aakhupashana - - - + -Darumocha - - - + -Darumoosha + - - - -Darumooshaa - - - + -Gauripashana + - - + -Hatachoornaka + + + - -Malla + - + - +Mallaka - - - + -Mushaka + - - - -Peeta - - - + -Phenashma - - - + -Phenashmabhasma - - - + -Sambala + - - + -Sankhiya - - + - -Shankhamusha - - - + -Shankhavisha + - + + -Somala + - + + -Sumbalakhara - - + - -Talasatwa + - - - -Ullipashana - - - - +Vikata + + + - - 21
    • Table No. 2: Shows the types of Malla based on colour. Reference Shweta Rakta Peeta Krishna 18 A.P + - + - R.T19 + + - - Y.R20 + + - - B.R.R.S21 + - + +Table No. 3: Shows the types of Malla based on appearance.Reference. Sphatik- Shankh- Haridr- Dadim- Shwet- Vikata. Hata- -abha. -abha. -abha. -abha. -abha. -Choor- -naka.R.R.S22 + + + - - - -A.P 23 - + - + - - -Y.R 24 - + - + - - -R.C 25 - - - - - + +R.J.N26 - + + - - + +Based on occurrence8:Natural/ Khanija/ Parvata sambhava and Artificial/ Krutrima.All the shwetha varieties are krutrima. Rakta, peeta are parvata sambhava.Need for Malla Shodhana: As Malla is considered as visha27,Aakhupashana,Shankhavisha, Dhatuvisha28, and now a days it is prepared and availablein artificial form it should be subjected for Shodhana prior to its internal use as medicine.Malla shodhana: As per different texts there are different Medias indicated.Table No. 4: Shows the shodhana of Malla according to various authors.S.N. REF PROCEDURE DRUGS USED METHOD DURATION 1. R.T 29 Pachana Karavellaka swarasa Dola yantra 6hrs 2. R.T 30 Pachana Meghanada swarasa Dola yantra 1 day 3. R.T 31 Pachana Aja dugdha/Ajarasa Dola yantra 1 day 4. R.T 32 Pachana Tankanajala Dola yantra 3hrs 5. R.T 33 Pachana Godugdha Dola yantra 3hrs 6. Y.R34 Pachana Meghanada swarasa Dola yantra 1 day 7. Y.R35 Pachana Kanji/Tankanajala/ Dola yantra 6hrs 22
    • Ajamamsarasa/ Godugdha. 36 8. Y.R Putapaka Kadalikanda Puta paka 6hrs 9. R37 Pachana Godugdha/ Dola yantra 6hrs Ajadugdha/ Karavellaka swarasa.10. R.R.S38 Swedana Karavellaka swarasa Dola yantra --11. R.C39 Swedana Karavellaka swarasa Dola yantra 4hrs12. R.C40 Swedana Ghananada Swarasa Dola yantra 4hrs13. R.C41 Swedana Kanji/Tankanajala/ Dola yantra 2 Ghatika Ajamamsarasa/ Godugdha.14. R.C42 Putapaka Kadalikanda Putapaka 2 Ghatika15. R.C43 Pachana Mahishajala/ Dola yantra 1Yama/ Mahisha dugdha 1Yama16. R.J.N44 Swedana Karavellaka swarasa Dola yantra 1YamaMalla Marana: 45,46,47,48 1) Take a wide mouthed vessel and keep 2 Palas Suryakshara in it. Above that,place 2 Palas of Hastidanta churna in such a way that it should look like a heap (i.e. wideat base and sharp at the tip). Keep this vessel on oven and heat it. When both the drugsget mixed up and there is no smoke, fire is extinguished. Take out the mixture, mix itwell and preserve it. Take 2 Tolas of this in a Moosha and place 1 Tola Shuddha MallaKhanda over it. Again it is covered by 2 Tolas of above mixture. Mukhabandhana is doneby using a Sharava and heat is given in a Lavaka Puta. By this white Malla Bhasma isprocured. 2) Take 3 Tolas Rumimastagi Churna. Take one mud pot and keep half (11/2Tola)of Rumimastagi Churna in that. Above that keep 1Karsha Shuddha Malla and above thatfill the remaining Rumimastagi Churna. Cover that mud pot by using an iron vessel anddo Sandhibandhana.. Immerse the mud pot in Arka dugdha and heat it for 1Prahara. Bythis we will get Malla Bhasma. 23
    • 3) Take white ash of Kantharikashta. Take one mud pot. Fill half of it with ashesof Kantharikashta. Keep 1Karsha Shuddha Malla above that. Remaining Kantharikashtaashes are then put over it. Keep that pot on oven and Badarakashtagni is given for 2Yamas. After self-cooling collect the Malla Bhasma. 4) Take Shuddha Malla in a mud pot. Put 5 Karshas of Ajadugdha above that. Itis covered by a cloth. Place this in a pit and cover it by 1Angula mud. Ignite 10Vanopalas of agni. Like this the procedure is repeated for 21 times. For each Puta, add 5Tola Ajadugdha. By this we will get Peeta-Aruna coloured Malla Bhasma.Satwapatana: 49, 50, 51 Take equal parts of Shuddha Malla and Saurashtri churna. Both are keptin a mud pot. Add 20 Tolas Rambhatoya to it. Place one more mud pot on it and dosandhibandhana. Keep it on oven and heat it for one Prahara. After self-cooling collectthe material deposited on the upper pot. It is indicated in Shwasa, Kasa, and Jwara. It canbe given along with Ghrita and Sita, Sheeta jala, Dadhi, Dadhyanna. Malla satwa is prepared as that of Haratala satwa. Malla satwa is Shubhravarnatmaka. Essence of Malla is similar to that of Haratala. The essence is pure White,soothing and destroyer of the three doshas. It is used in the solidification of Mercury andincreases the potency of the same.Pharmaco-therapeutic properties:Table No. 5: Shows Gunas of Malla according to various Rasa classics. SL.NO GUNA R.R.S 52 R.T 53 D.G.V 54 1 Snigdha + + - 2 Laghu - - + 3 Rooksha - - + 4 Teekshna - - +Rogaghnatha: 55 Destroys kapha vata predominant diseases, and also useful in scorpion bite,Shwasa, Kasa, Kushta, Shleepadottha Jwara, Yakshma, Sandhivata, Phiranga,Agnimandya, Vishamajwara, Jeerna Pandu, Pratamaka Shwasa, Hrudshoola,Hruddaurbalya, Atisara etc. Externally it acts as Kshara. It is Shotha-Santapa-Shaithilya 24
    • nashaka.When Malla applied to the region of scorpian bite along with water; it kills thepain in ½ Ghati 56. Shuddha Malla is Balya, Vrushya, Rasayana, Shwasa, Sheeta Jwara, andPandunashaka. It is also useful in Pleehavruddhi, Phiranga, Shleepada, Kushta andSandhivata57.It is useful in Vata-Kapha-Sheeta Vyadhis58.Preparation of Gauripashana (Malla): 59 Haratala rubbed with Castor oil (Eranda Taila) and Lime juice (Nimbu rasa)and heated by means of a Valuka Yantra, exhibits Red Gauripashana as itsessence.Matra: 1/120th –1/30th of Ratti.60 1/10th – 1/3rdSarshapa.61Matra Nirmana Vidhi: 62 Take 1 Gunja – Shuddha Malla. 15 Masha – Maricha Churna. Q.S. – Shrungavera Swarasa. Take Shuddha Malla and Maricha Churna in a Khalwa and triturate withAardraka Swarasa for 3 days. Prepare 1 Ratti pramana Vatis. 1 vati in the morning andone in night is given for dosha shamana. In this quantity itself it is added to otherformulations and is used.Care during Matra Nirmana63: 1 Ratti Malla causes death. So according to Desha, kala, rogi bala, doshaetc matra is decided and given. Fatal Dose: 1 Ratti (125mg).Aamayika Prayoga: 64 Along with various Anupana’s Malla is indicated in various diseases. Vasa Satwa/ Vyaghri churna Purana Shwasa. Panchatikta Satwa/churna Kushta. Guduchi churna/Arka churna Shleepada Jwara. Karanjabeeja churna Cures Vishama Jwara in one day. 25
    • Shunthi and Punarnava churna Amavata and Amavataja Jwara. Loha Bhasma with Triphala Kashaya Purana Pandu. Jatipatra and Lavanga churna Purana and Daruna Phiranga. Rasasindhura and Loha Bhasma Rajayakshma. Shankhapushpi churna Hruddaurbalya. Shunthi and Maricha churna Bhuktamatra samudbhava Atisara. Malla triturated with Jala- Lepa Vruschikadamsha Vishanasha.Pathya: 65 Milk, Sugar, Butter, grams and other nutritious substances should be taken in large quantities. 66Apathya: Salt, chillies, oil, asofoetida and other hot and spicy things should beavoided. Vishishta yogas: ♦ Malla Sindhura. ♦Sameerapannaga Rasa. ♦ Suchikabharana Rasa. ♦Malla Vati ♦ Shankhavishodaya Rasa. ♦ Malla Chandrodaya rasa ♦ Kalanala Rasa. ♦ Malla Garbha Pottali. ♦ Chandeshwara Rasa. ♦ Panchasuta ARSENICArsenic is ordinarily presumed to be a metal, is in fact a metalloid.67Arsenic68 is found in the free state but it occurs more commonly in combination withiron as arsenical iron pyrites or mispickel, FeS2, FeAs2 and as arsenical iron orleucopyrite, FeAs2.Arsenic forms two oxides-arsenic trioxide,As2O3(the anhydride ofarsenious acid,H3AsO4) and arsenic pentoxide,As2O5(the anhydride of arsenic acid,H3AsO4).The trioxide is the most important compound of arsenic.Arsenic69 is found in nature at low levels. It’s mostly in compounds with oxygen,chlorine, and sulfur (inorganic arsenic compounds). Arsenic in plants and animalscombines with carbon and hydrogen (organic arsenic). Organic arsenic is usually lessharmful than inorganic arsenic. Most arsenic compounds have no smell or special taste.When arsenic enters the environment: It doesn’t evaporate. Most arsenic compounds can 26
    • dissolve in water. It gets into air when contaminated materials are burned. It settles fromthe air to the ground. It doesn’t break down, but can change form. Inorganic arsenic is ahuman poison. Organic arsenic is less harmful. Arsenic damages many tissues includingnerves (peripheral polyneuropathy, axonal degeneration), stomach and intestines, andskin. All arsenicals except arsine act by inhibiting sulfhydryl enzyme systems requiredfor cell metabolism and the potency of action depends on the valence of the arsenic atom.Arsine reacts with hemoglobin to form a very strong hemolytic poison.Lower levels of exposure to inorganic arsenic may cause nausea, vomiting, anddiarrhoea, decreased production of red and white blood cells, abnormal heart rhythm,blood vessel damage, a “pins and needles” sensation in hands and feet, painful andprofuse diarrhoea, shock, coma, convulsions and death, irritation, inflammation,ulceration of mucous membranes and skin, kidney damage. Direct skin contact may causeredness and swelling.Chronic toxic effects69: Fatigue, loss of energy, G.I. disturbance, nasal septumperforation, ulceration in folds of skin, increased pigmentation of skin, appearance ofsmall “corns” or “warts” on the palms, soles, and torso, exfoliative dermatitis, rashes,muscular paralyses and atrophy, sensory disturbances, visual disturbances and blindness,degeneration of liver (cirrhosis) and kidneys, garlic odour to breath, non cirrhotic portalhypertension. Arsenicosis is a chronic disease due to drinking water containing arsenic.Arsenic is an ultra-trace essential element (nonmetal) 70: The deficiency signs are impairment of growth, reproduction, heart function.The specific function is increased arginine leads to urea and ornithine which helps inmetabolism of methyl compounds71. Arsenic affects arginine, membrane phospholipidsand zinc metabolism. It is less toxic than Selenium, an ultra trace element with anestablished role72. Selenium in toxic amounts is corrected by Arsenic which has a positiveeffect. 73The order of toxicity of arsenic compounds is74 :Arsines (As (III))>arsenite (As(III))>arsenate (As (V)) and arsenic-organic acids (As (V)).Arsenic which is foundmainly in liver,lungs,kidneys and intestinal walls, is readily absorbed if watersoluble.Arsenic is also toxic by replacement of Phosphorous in ATP readily absorbed ifwater soluble.Lethal dose of more toxic forms of arsenic is 125 mg per kg body weight. 27
    • But for less toxic forms, say arsenic in drinking water, larger quantities arenecessary, i.e, 100-200 mg per kg body weight. An antidote for arsenic is BAL, BritishAnti Lewisite.74The toxic effect of arsenic trioxide is by attacking –SH groups of an enzyme, therebyinhibiting enzyme action75.SHEnzyme + As-O Enzyme As-O+2OHSHFig 1: Showing the As and enzymatic action.Compounds of Arsenic76: In the early part of the present century Ehlrich began theinvestigation of organic arsenic compounds, with the object of preparing substanceswhich might be of the value in the treatment of spirochetes or protozoan diseases such assyphilis ,yaws, relapsing fever ,sleeping sickness, and amoebic dysentery. The realproblem was to obtain a substance which would be highly active in exterminating theparasites, and yet would have as low toxicity as possible to the host. As a result of a greatdeal of research, a number of arsenical compounds were selected that had these propertiesto a high degree. They proved of great medicinal value, especially in the period beforethe introduction of antibiotics.Haematology: 77 Serum Arsenic level is 7 mg/ dl. Urinary excretion of Arsenic 100mg/day or more indicates arsenic poisoning. 78PotencyThe LD50 for pure arsenic is 763 mg/kg (by ingestion) and 13 mg/kg (by intraperitonealinjection). For a 70 kg (~155 lb) human, this works out to about 53 grams (less than 2ounces). However, compounds containing arsenic can be significantly more toxic. 28
    • ARSENIC TRIOXIDE Arsenic trioxide which was official in all earlier pharmacopoeias, is thecommonest compound of arsenic, and is obtained as a byproduct in many metallurgicaloperations, particularly in the extraction of tin, nickel,and cobalt. At least three forms ofarsenic trioxide are known, and the ordinary substance is a mixture of an amorphousvitreous form and an octahedral form.79 HISTORY OF ARSENIC TRIOXIDE (AS2O3)80 Because Of its significant medicinal properties, arsenic has been used astherapeutic agent since 2,400 years .In the 15th century William Withering whodiscovered digitalis was a strong proponent of arsenic-based therapies. He argued“Poisons in small doses are best medicines; and the best medicines in too large doses arepoisonous. Pharmacology texts of 1880’s describe the use of arsenical pastes for cancersof skin and breast and arsenous acid was used to treat hypertension, bleeding gastriculcers, heart burn , chronic rheumatism.Arsenics reputation as a therapeutic agent wasenhanced when Noble laureate Paul Ehlrich developed ‘Salvarsan’ an organic arsenical,for treating Syphilis and Trypanosomiasis.Arsenics antileukemic activity was reported inlate 1800,s.In 1878 a report from Boston City Hospital described the effect of Fowlerssolution on reduction of white blood cell counts in two normal people and one patientwith leucocythemia.Subsequently Arsenic trioxide was administered as a primaryantileukemic agent until it was replaced by radiation therapy. In one report from Chinathe monotherapy of Arsenic trioxide produced a complete clinical response in 9 of 10with relapsed APL.Consequently, TRISENOX (As2 O3) was approved for the relapsed orrefractory APL by US FDA in September 2000.Arsenic trioxide81 was the active ingredient of the liquor arsenicals or arsenical solutionof 1932, 1948 and 1953 pharmacopoeias. The preparations were widely prescribed intonics.Other names :- Arsenic(III) oxide Arsenolite Arsenic sesquioxide Arsenicum album Arseneous oxide Arseneous anhydride 29
    • Properties: Arsenic trioxide is a white or transparent solid in the form of glassy,shapeless lumps or a crystalline powder that resembles sugar. It has no odor or taste. Itforms readily when elemental metallic arsenic is heated to high temperatures or burned. Physical and Chemical properties82 Molecular formula As4O6,As2O3 Molar mass 197.841 g/mol Fig 2: Arsenic trioxide Appearance : White solid Opaque mass. Odor : No Odor. Taste : No taste/ slightly metallic taste. Hardness : 3–4 Specific gravity : 5.6 – 5.8 Molecular weight : 197.84 Daltons Boiling point (760 mm Hg) : 869ºF (465ºC) Sublimes at : 379ºF (193ºC) Melting point : 594ºF (312ºC) Vapor pressure : 66.1 mm Hg at 594ºF (312ºC) Density and phase : 3.86 g/cm³, solid. Solubility in water : 2 g/100 ml (25°C) Acidity (pKa) : 9.2 Mohs hardness : 1.5 Refractive Index : 1.755 Lattice constant : 11.074 Å Crystal structure : Cubic (α) <180°C Monoclinic (β) >180°C Dipole moment : Zero Specification : As2O3-99.5%,Fe-0.05%,moisture-0.5% Standard enthalpy 30
    • of formation ∆fHosolid : −657.4 kJ/mol Water solubility: Low solubility in water (37 g/L at 20ºC, 115 g/L at 100ºC); slightly soluble in alcohol; soluble in dilute HCl solutions. Flammability: not flammable, but emits highly toxic arsine gas and oxides of arsenic fumes when burned.Arsenious oxide on sublimation forms a transparent glassy mass which graduallybecomes opaque in presence of moisture. It is a colorless, odorless, taste less substance83. Arsenic trioxide is an amphoteric oxide which shows a marked preponderance forits acidic properties. It dissolves readily in alkaline solutions to give arsenites. It is muchless soluble in acids, but will dissolve in hydrochloric acid to give arsenic trichloride orrelated species. It reacts with oxidizing agents such as ozone, hydrogen peroxide andnitric acid to give arsenic pentoxide, As2O5: the reaction with hydrogen peroxide can beexplosive. It is also readily reduced to arsenic, and arsine (AsH3) may also be formed. Related Compounds: Other anions: Arsenic Trisulfide Other Cations: Phosphorous trioxide, Antimony trioxide.Varieties84: Vitreous amorphous form: Specific gravity 3.74 which is more or less transparent and melts without volatization at approximately 2000C. Crystalline octahedral state: Melting point 2750C and specific gravity 3.69. It sublimates without fusion. Monoclinic variety : Melting point 3150C and specific gravity 3.85. In industries AS2O3 is purified by repeated sublimations in iron pots. Aprecaution is taken that the AS2O3 is not reduced to As, which would then eat up the ironpots and more over it would fall into furnace with giving poisonous gases85.Tests86 The following are the main tests of arsenic. Arsenic compounds when heated on charcoal give a white encrustation far from the assay and at the same time fumes having a garlic odor are emitted. 31
    • Heating in the open tube arsenic compounds gives a white sublimate which is volatile on heating Heated in the closed tube some arsenic compounds give shiny black sublimate the arsenic mirror; most arsenates give a similar mirror when heated with charcoal or sodium carbonate in the closed tube.Pharmacological actions87: Organic arsenicals used in chemotherapy of trypanosomiasis, amoebiasis andtrichomoniasis in the form of carbarsone, tryparsomide, cycobiarsol, melarsoprol. Theyare no longer in the treatment of syphilis.Pharmacology: 88 1. Inorganic arsenicals used mainly as rodenticides, herbicides and insecticides. 2. Organic arsenicals in the chemotherapy of Trypanosomiasis. They were in the mainstay of treatment of syphilis.Absorption, fate and excretion: 89 The inorganic forms of arsenic are more toxic than the organic forms and thetrivalent forms are more toxic then the pentavalent forms. Arsenic can be inhaled,absorbed through the skin, or absorbed in the GI tract after ingestion. After a very smalldose of arsenic (like those experienced daily by most people) most of the absorbedinorganic arsenic undergoes methylation, mainly in the liver, to monomethylarsonic acidand dimethylarsinic acid which are excreted, along with residual inorganic arsenic in theurine. However, if the dose of arsenic is very large, the elimination half-life isprolonged.Once absorbed, arsenic rapidly combines with the globin portion ofhaemoglobin and therefore localises in the blood. There is minimal penetration of theblood-brain barrier, and within 24 hours arsenic redistributes itself to the liver, kidney,spleen, lung and GI tract, with lesser accumulation in muscle and nervous tissueToxbase summary 90Toxicity: Generally less acutely toxic than soluble arsenic salts. A patient has died after ingesting 2 g. Death from acute arsenic poisoning is usually caused by irreversiblecirculatory insufficiency, but if the dose is not large enough to kill the patient a number 32
    • of secondary effects can be seen 2-4 weeks after ingestion of the poison. These includehair loss, Mee’s lines (white transverse lines seen on the nail plate up to a year afterarsenic intoxication), sensorimotor peripheral neuropathy (may develop within a fewhours of ingestion but usually seen 2-8 weeks after exposure), skin changes (as in chronicpoisoning) and possible chronic renal failure.Fatal dose91: 200 – 300 mg.Fatal period91: 12-48 hrs. The shortest period is 45mins.Prehospital Management92 Quickly assess for a patent airway, and ensure adequate respiration and pulse. Maintain adequate circulation.Skin Exposure: Wash exposed skin and hair with mild soap and water, and rinsethoroughly with water. Use caution to avoid hypothermia, particularly with children andthe elderly.Eye Exposure: Flush exposed or irritated eyes with plain water or saline for at least 15minutes. Remove contact lenses if easily removable without additional trauma to the eyeIngestion: Do not induce emesis. The effectiveness of activated charcoal is questionable,but administration of activated charcoal as an aqueous slurry in persons who are awakeand able to protect their airway is recommended pending further evaluation in cases ofingestion of unknown quantities. Activated charcoal is most effective when administeredwithin 1 hour of ingestion. At 1 gram per kilogram (gm/kg), the usual adult dose is 60-90grams (g), and the child dose is 25-50 g. A soda can and straw may be of assistance whenoffering charcoal to a child. Complications include emesis and aspiration. Persons with evidence of significant exposure and all persons who have ingestedarsenic trioxide should be transported to a medical facility for evaluation.Hospital/Emergency Room Management 33
    • Evaluate and support the airway, breathing, and circulation as appropriate. Establish intravenous access in symptomatic patients and monitor cardiac rhythm.Antidote93: Hemodynamic stabilization and gut decontamination are key factors in theinitial management of acute arsenic intoxication. Chelating agents administered withinhours of arsenic absorption may successfully prevent the full effects of arsenic toxicity. 1. Dimercapol /BAL 2. Freshly prepared gelatinous hydrated ferric oxide is given which acts with arsenious trioxide to form ferric arsenite and it is a harmless salt. 3. DMSA (succiner) – Meso 2,3 Dimercapto succinic acid 4. DMPS (Unithiol) – 2, 3 Dimercapto propane – 1 Sulfonate. Both 3 & 4 decreases the arsenic content in the tissue.Arsenic trioxide uses94: • Starting point for the manufacture of arsenic-based pesticides. (Sodium arsenite, sodium arsenate, sodium cacodylate). • Starting point for the manufacture of certain arsenic-based pharmaceuticals (Neosalvarsan) and veterinary products. • Decolorizing agent for glasses and enamels and Preservative for wood. • Hydrogen recombination poison for metallurgical studies. • Starting point for the preparation of elemental arsenic, arsenic alloys and arsenide semiconductors. • Use as a cytostatic in the treatment of refractory promyelocytic (M3) subtype of acute myeloid leukemia. The drug is available as Trisenox® ampules; each containing 10mg to be diluted for i.v. infusion. • Medical applications: Arsenic trioxide under the trade name Trisenox (manufacturer: Cephalon) is a chemotherapeutic agent of idiopathic function used to treat leukemia that is unresponsive to first line agents. It is suspected that arsenic trisulfide induces cancer cells to undergo apoptosis. Due to the toxic nature of arsenic, this drug carries significant risks. • The enzyme thioredoxin reductase has recently been identified as a target for arsenic trioxide. 34
    • KARAVELLAKAINTRODUCTION The drug Karavellaka (Momordica Charantia) belongs to Shaaka Varga,Cucurbitaceae family is seen throughout in India and also in Malaya, China, tropicalAfrica and America, up to an altitude of 1500m. . It is a Climber bearing yellow coloured flowers. It is cultivated in gardenseverywhere in India for its fruits. It is used as vegetable. Fruit is bitter in taste. Fruitsbear numerous triangular tubercles like Crocodile’s back. All parts of this plant can beused.HISTORY: Karavellaka is mentioned in all Brahattrayis and almost all Nighantus. It is usedby Unani Hakim’s also.SAMHITA PERIOD: Karavellaka is mentioned under Tikta Skandha95. Karavellaka is indicated for Vrana.96 and can be used to improve vision.96 . Inclusion of Karavellaka in Aragvadhadi Gana97 . Karavellaka is also included in Tikta Varga97. Karavellaka is one of shaakhas which is having bitter taste and mitigates Kapha and pitta98NIGHANTU PERIOD 99,100,101,102,103 We find the reference of Karavellaka and its qualities in : 1) Adarsha Nighantu 4) Kaiyadeva Nighantu 2) Bhavaprakasha Nighantu 5) Raja Nighantu 3) Madanapala NighantuNIRUKTI104 “Kaarena prayatnena yashtyadikamavalambya vellati chalati iti Karavellaha Vellam chalane│ Karam jwaradinadyam Prativellati iti” It motivates all its sources/destroys diseases like Jwara. NOMENCLATURE: Sanskrit name Karavellaka Latin name Momordica Charantia 35
    • Kingdom Plant Kingdom Class Dicotyledons Sub-Class Polypetalae Series Calyciflorae Order Passiflorales Family Cucurbitaceae Genus Momordica Species Charantia Kula Koshataki KulaGana: Sushrutokta Gana Aragvadhadi GanaVarga: Sushrutha Samhitha Tikta Varga Charaka Samhitha Shakha Varga Astanga Hridaya Shakha VargaSYNONYMSTable No: 6 Shows Synonyms of Karavellaka.SYNONYMS A.N105 M.N106 B.N107 R.N108 K.N109Brihatvalli - + - - -Kandakatuka - - - + +Kandeera - - - + +Karavella - + + - -Karavellaka + - - - -Karavelli - + + + -Kathila - - + - +Katila + + - - -Noyavalli - - - + -Patu - - - + - 36
    • Sukanda - - - + +Sushavee + - - - -Ugrakanda - + - + +Varivalli - + - - -VERNACULAR NAMESSanskrit Karavellaka, Pitapushpa, Sushavi, Toyavalli, Ambuvallika, Chiripatra, Brihadvalli, Padu, Karavalli, Sukandaka, Katilla.Arabic QisaulbarriAssam Kakiral,KakralBengali Barmasiya, Jethuya, Karala, ucheteEnglish Carrilla fruit, bitter gourdGujarati Karela, Karelo, KareluHindi Karela, kareli, KarolKannada HagalakayiKonkani KaratimMalayalam Kaippa, Kaipavalli, Pavakka ChetiMarathi Karalu,KarliOrissa Kalara,SalaraPunjabi Karela, KarilaSimhalese Karawila, BattukarawillaTamil Pavakkay, Paval, pakarTelugu KakaraTulu KanchalaUrdu KarellaSCIENTIFIC CLASSIFICATION 110: Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Cucurbitales Family: Cucurbitaceae 37
    • Genus: Momordica Species: M. charantiaSynonyms110: Momordica chinensis, M. elegans, M. indica, M. operculata, M. sinensis,Sicyos fauriei.Description:- 111 Momordica charantia is a tropical and subtropical vine of the familyCucurbitaceae, widely grown for edible fruit, which is among the most bitter of allvegetables. English names for the plant and its fruit include bitter melon or bitter gourd. The herbaceous, tendril-bearing vine grows to 5 m. It bears simple, alternateleaves 4-12 cm across, with 3-7 deeply separated lobes. Each plant bears separate yellowmale and female flowers.The fruit has a distinct warty looking exterior and an oblongshape. It is hollow in cross-section, with a relatively thin layer of flesh surrounding acentral seed cavity filled with large flat seeds and pith. Seeds and pith appear white inunripe fruits, ripening to red; they are not intensely bitter and can be removed beforecooking. However, the pith will become sweet when the fruit is fully ripe, and the pithscolor will turn red. The pith can be eaten uncooked in this state, but the flesh of the melonwill be far too tough to be eaten anymore. The flesh is crunchy and watery in texture,similar to cucumber, chayote or green bell pepper. The skin is tender and edible. The fruitis most often eaten green. Although it can also be eaten when it has started to ripen andturn yellowish, it becomes more bitter as it ripens..Bitter melon comes in a variety ofshapes and sizes.Main Actions 111 Other Actions111 Standard Dosage111 Kills bacteria reduces inflammation Kills viruses’ fights free radicals Decoction: 1 cup 1-2 times daily Kills cancer cells enhances libido Tincture: 1-3 ml twice daily Kills leukemia cells cleanses blood Capsules: 1 g twice daily prevents tumors detoxifies Treats diabetes expels worms reduces blood sugar balances hormones reduces blood pressure enhances immunity lowers body temperature mildly laxative Lowers cholesterol promotes milk flow 38
    • Pharmacological activities 112:Antispasmodic, antioxytocic ,hypoglycemic ,mild cholinergic blockingactivity,abortificent,antipyretic,mild-hypotensive,anti-bacterial,uterinestimulator,anthelminthic,insecticidal,anti-viral,anti- lipolyticantidiabetic,antimalarial,antispermatogenic,antisteroidogenic,androgenic,anti-ulcerogenic,antifungal,juvenomimetic.PROPERTIES :Table no 7: shows the Pharmacological Properties of Karavellaka. RASA GUNA VEERYA VIPAKAKaiyadeva Nighantu113 Tikta, Katu - - KatuDravyaGuna114 Tikta, Katu Laghu, Rooksha Ushna Katu 115Raja Nighantu Katu, Tikta - Ushna -Astanga Hridaya116 Katu,Tikta - Ushna KatuAdarsha Nighantu117 Tikta - Sheeta KatuDOSHAGHNA KARMAThe drug is said to be, Pittashamaka, Kapha Pittashamaka, TridoshashamakaROGAGHNA KARMA♦ Madhumeha nashaka ♦ Shothahara♦ Jwaraghna ♦ Vishaghna♦ Kushtahara ♦ Vranaghna♦ Krimighna ♦ Netra roga♦ Arsha ♦ Karna shoola♦ Pittaja mastishka shoola ♦ Visuchika♦ Nadivrana ♦ Visarpa♦ Pandu ♦ Vatarakta♦ Kamala ♦ Shwasa♦ Kasa ♦ Shleepada♦ Galaganda ♦ AmlaPitta 39
    • AUSHADHA MATHRA The dose of the Karavellaka according to its different forms is as follows :- Swarasa - 10-30 ml Choorna - 3-6 masha Including emesis- 100mlFor Madhumeha - 3-6gms of Choorna of fruit with waterFor Ashmari - 3 tola of leaf Juice.AMAYIKA PRAYOGAEXTERNALLY • The whole plant is applied in scabies and other cutaneous diseases, in combination with the oil of Cinnamon etc. • In Leprosy, fruit is used. • In Night blindness - juice of leaves applied around the orbit with black pepper. • In burning of soles - juice of leaves is rubbed to soles 2 or 3 times a day.INTERNALLY • It acts as stomachic tonic, anthelmintic, Rheumatism, gout, fruit is used. • Anthelmintic - fruits and leaves are used. • Emetic, purgative - juice of leaves with purgatives. • It is also used in Jaundice, as an appetizer, blood purifier, dysmenorrhea, urinary disorder, asthma, cough, obesity etc. • In diabetes-juice of raw fruit, ½ ounces at a time, once daily in empty stomach early in morning. PHYTO CHEMISTRY118: Bitter melon contains an array of biologically activeplant chemicals including triterpenes, proteins, and steroids. One chemical has clinicallydemonstrated the ability to inhibit the enzyme guanylate cyclase that is thought to belinked to the cause of psoriasis and also necessary for the growth of leukemia and cancercells. In addition, a protein found in bitter melon, momordin, has clinically demonstratedanticancerous activity against Hodgkins lymphoma in animals. Other proteins in theplant, alpha- and beta-momorcharin and cucurbitacin B have been tested for possibleanticancerous effects. Two of these proteins-alpha- and beta-momorcharin-have also beenreported to inhibit HIV virus in test tube studies. In one study, HIV-infected cells treated 40
    • with alpha- and beta-momorcharin showed a nearly complete loss of viral antigen whilehealthy cells were largely unaffected. The chemicals that lower blood sugar include amixture of steroidal saponins known as charantins, insulin-like peptides, and alkaloids. 118Its Constituents : Alkaloids, charantin, charine, cryptoxanthin, cucurbitins,cucurbitacins, cucurbitanes, cycloartenols, diosgenin, elaeostearic acids, erythrodiol,galacturonic acids, gentisic acid, goyaglycosides, goyasaponins, guanylate cyclaseinhibitors, gypsogenin, hydroxytryptamines, karounidiols, lanosterol, lauric acid, linoleicacid, linolenic acid, momorcharasides, momorcharins, momordenol, momordicilin,momordicins, momordicinin, momordicosides, momordin, multiflorenol, myristic acid,nerolidol, oleanolic acid, oleic acid, oxalic acid, pentadecans, peptides, petroselinic acid,polypeptides, proteins, ribosome-inactivating proteins, rosmarinic acid, rubixanthin,spinasterol, steroidal glycosides, stigmasta-diols, stigmasterol, taraxerol, trehalose,trypsin inhibitors, uracil, vacine, v-insulin, verbascoside, vicine, zeatin, zeatin riboside,zeaxanthin, and zeinoxanthin are all found in bitter melon.Physical Constituents in the Fruit: 119Foreign matter-NilTotal Ash-Not more than 8.5%Acid-insoluble Ash-Not more than 0.6%Alcohol soluble extractive- Not less than 6%Water –soluble extractive-Not les than 28%Dosage and administration 118 : Bitter melon may be consumed in the following ways: as food, a decoction of asmuch as 3 1/3 ounces (100 ml), or 2 ounces (60 ml) of fresh juice (daily). Though stillbitter, tinctures of bitter melon (1 teaspoon [5 ml] two to three times per day) may also beused. The above dosages would be appropriate for diabetics.Side effects and interactions 118: Excessive ingestion of bitter melon juice (several times more than the amountrecommended above) may lead to diarrhoea and abdominal pain. Consuming excessiveamounts of the seeds may be linked with fever, headache, and coma. Pregnant womenshould not use bitter melon. Hypoglycemia, or low blood sugar, may be negativelyaffected by bitter melon because it may trigger or worsen the condition. 41
    • VISHISHTA YOGAS:- ♦ Sheeta nagankuram ♦ Jwarari rasa ♦ Kacchuradi taila ♦ Brihath Vishama jwarantaka lauha ♦ Maha vishagarbha taila ♦ Vidya vallabha rasa. 42
    • PHARMACEUTICAL REVIEW ‘Kushala Rasa Karmani’ is the quality of a RasaShastraghna120.Theintegral Part of Rasashastra lays in the successful pharmaceutical process .Among distinctSamskaras like Shodhana, Marana, and Jarana etc. Shodhana stands par excellence.So here is a gross view on the Swedana process of the drug. . In a broader sense theabove said basic pharmaceutical procedure can be denoted under the heading of theprocedures adopted and the Yantras used in the study. Practically the above saidprocedure will be carried out and hence to aid the theoretical clarity a brief note has beendrawn vide infra.PROCEDURE ADOPTED:Samskara121: ‘Gunantharaadhaanam’ i.e enhancement of the qualities of a drugand ‘Doshaapanayanam’ i.e to remove the unwanted impurities. One has to applyVarious Techniques such as122 • Toya Sannikarsha- to the vicinity of water. • Agni Sannikarsha- to the vicinity of fire. • Toyagni sannikarsha-to the vicinity of both fire and water. • Shoucha- to wash or cleanse. • Manthana- to Churn. • Bhajana-by using the utensils. • Desa- the place/region of preparation. • KalaPrakarsha- time period taken. • Bhavana- by Trituration.Shodhana123: “Shodhayati iti” (Shudh +nich+pluth) That which purifies,cleanses and makes Pure. 124Need for Shodhana : Just like a cloth that cannot be coloured until it is washed orcleansed.In the same way the process of purification is a must in case of raw drugs. Shodhana 125,126,127 is the pharmaceutical procedure in which all the drugs oforigin from metals , minerals, animals and herbs are subjected, before subjecting them toMarana or before administration in case of some Rasa Dravyas like Malla, Shilajatu,Gairika, Kasisa etc. 43
    • The literal meaning of shodhana is purification. But in Rasashastra, shodhana isnot merely purification, but is a samskara, which essentially brings out modifications oralterations in properties along with purification.Historical Background: Bruhathrayis do not mention about the special methods of shodhana of metalsand minerals. It is only in the golden era of Rasashastra and Nighantu period dealtelaborate description of shodhana of minerals and metals as well as herbal drugsspecifically. Most of the raw materials in Rasashastra are native from earth. So the very chanceof impurities, toxicity, heterogeneous qualities, mixing of other substances and unwantedqualities to a large extent. Now a days some of the Rasadravyas are artificially prepared.So shodhana is indicated to eliminate all such toxic qualities, induce and enhane certainspecial qualities which are essential for the easy assimilation of the material in the livingbody.Definition: 128,129,130 “Shodhanam karma vijneyam dravya dosha nivaaranam” The process which eliminates the blemishes is called shodhana. “Uddhishtairoushadhaihi saardham kriyate peshanaadikam Mala vichchittaye yattu shodhanam thadihochyate” When a substance is subjected to trituration etc with required medicinefor removal of unwanted materials or impurities is known as shodhana. “Loha dhatu rasaadeenamudithaihi aushadhaihi saha Swedanam mardanam chaiva tailado daalanam tatha Doshaapanuttaye vaidyaha kriyate shodhanam hi tat” Subjecting the Loha, Dhatu, Rasoparasas etc to the procedures like swedana,mardana etc with the prescribed medicines, Dhalana in tailadi dravadravyas to removethe Doshas is termed as shodhana.The meaning of shodhana can be:♦ To Clean ♦ To Dehydrate 44
    • ♦ To Distil ♦ To Polish♦ To Peel ♦ To Dehusk♦ To Clarify ♦ To Filter♦ To Wash ♦ To PurifyObjectives of Shodhana: • To make metallic substances suitable for Marana. • To remove Physical and Chemical impurities & Regulation of Physico- chemical attributes. • Separation of admixtures & Elimination of harmful matters from the drug. • Metals are made free from blemishes. • Reduce or minimize toxic effect. • Make metal or mineral soft and brittle. • Reduction in particle size. • Make substance suitable for further processing. • Transformation of attributes or imbue organic qualities to inorganic substances. • Increasing the potency of the drugs. • Modification of undesirable physical properties of the drug. • Conversion of some of the characteristics of the drug to different stages. • Enhancement of therapeutic action. • Conversion of drugs from heterogeneous state to homogenous state. • Corrects the imperfections.Types of shodhana: 1. Samanya shodhana 2. Vishesha shodhanaSamanya shodhana: The common method used to purify a group of drugs is known asSamanya shodhana. This process eliminates general impurities of metals and mineralsand converts them into powder which is essential for further process. Ex: Samanya shodhana of dhatus by repeatedly quenching various liquid media.Vishesha shodhana:It is done specifically for a particular drug with the view ofpurifying it with the help of particular or specific shodhana material as well as procedure. 45
    • Ex: Swedana of Haratala in Kushmanda Swarasa by Dolayantra method.Various methods of Shodhana Various methods of shodhana are mentioned in Rasa classics. ♦ Swedana ♦ Prakshalana ♦ Mardana ♦ Bhavana ♦ Murchana ♦ Shoshana ♦ Patana ♦ Bharjana ♦ Avapa ♦ Samyoga ♦ Nirvapa ♦ Vibhaga ♦ Dalana ♦ Nimajjana ♦ Galana ♦ Pachana ♦ Dhavana. ♦ Washing ♦ Desumption ♦ Shifting ♦ Decoloration ♦ Bleaching ♦ Elutriation ♦ Boiling ♦ Maceration ♦ Pulverization ♦ Lixiviation ♦ Dipping ♦ Percolation ♦ Sublimation ♦ Dialysis ♦ Chemical combination These are also some of the methods of shodhana for many Rasadravyas. Thedravyas used might be kshara,amla,sneha etc.SHODHANA YANTRA: Different types of Yantras are mentioned in the Rasa classics for differentshodhana procedures. Viz Dolayantra,Damaru Yantra, Khalva Yantra, Urdhwapatanayantra, Sharava,Sthalika,Sandamsani, Darvi etc.Shodhana depends upon: Structure,Composition, Impurities, Qualities, Action of the drug.In the present study, for the shodhana of Malla, the Swedana method of shodhana hasbeen adopted as per classical text.Swedana:-Definition132: “Gharmodhgama roopa upakramam” “Samskaram mala shithila aapaadanaarthanam” The word ‘Swedana’ literally means causing to perspire, the act of sweating orperspiring. Boiling the drug by suspending in liquids (bathing) i.e. acids or alkalis or anymedicines, decoctions with the help of Dolayantra (a special apparatus for boiling) isknown as Swedana.133 46
    • “Kshaaramlairoushadhairvaapi dola yantre sthitasya hi Pachanam swedanaakhyam syanmalashaithilyakarakam”Uses: • Impurities soluble in acid or alkali are eliminated. • Mala shaithilyata. Different Yantras have been used for shodhana procedures and associated worksin the present study. They are 134 1. Dolayantra 2.Khalva yantra.Dolayantra 135,136,137: It is called Dola yantra because here the drug bundle to be subjected toSwedana is hung or suspended in a vessel containing liquid like a Dola (swing).A pottaliis prepared using three folds of cloth in which the drug is placed.Take a Pot and fill itwith the drava dravya prescribed to half of the pot . A strong rod should be placedtransversely after making small holes on either sides of the pot near its mouth. On thisrod, the pottali should be hanged with the help of a string till it is submerged in the liquidat the bottom of the jar. Ignite by placing the yantra over fire. Thereafter fomentationinitiates.Applied aspect: It is used to apply heat treatment to the drugs either through the boiling liquids orthrough the vapours for a specified time to loosen its impurities. The advantage ofapplying swedana by this method is to put the drug directly in contact with the boilingliquid which may help the drugs to be softened first and then allowing their solubleimpurities to be dissolved into the boiling liquids used for and thus making the drugs freefrom some of the impurities135. According to media used for Swedana, acidic, alkaline, volatile impurities geteliminated.Khalva Yantra : “yantram dronyaakaaram peshanopayogi yantram”138 It is a hollow, round or boat shaped apparatus made of iron, stone, glass or porcelain as per need. For mercurial operations, Khalvas made out of iron are preferred while for preparing pistis, bhasmas and formulations, shodhana of certain Rasadravyas Khalvas made out of stone are preferred.Generally Khalvas are of two types i.e. Vartula and Dronyakriti. 47
    • • Vartula Khalva is made of porcelain or stone. It should be 12 angula in radius, 4 angula in depth and 8 angula in length. • Dronyakriti or boat shaped Khalvas are generally used for mercury processing and made of iron or stone. Their height varies from 9” to 16”, length 16” to 24”, breadth 9” to 10”, depth 6” to 7” and thickness of their edges is 2” 139Uses: It is used for grinding, rubbing, triturating or mixing of drugs and liquids139.In the present study mortar and pestle made of porcelain were used for making smallpieces of Malla before subjecting to shodhana and after shodhana to make it a softpowder. ANALYTICAL REVIEW To evaluate the quality of the drugs, the scientific validation plays an importantrole. As a part of standardization, the classically processed Rasadravyas, preparedRasayogas will be subjected to mandatory baseline and current instrumental analysis. Inthe present study the two samples of Malla i.e. Ashuddha Malla and KaravellakaShodhita Shuddha Malla are screened for physicochemical and instrumentalstandardization methods. So the review of analytical procedures is carried out vide infra.Physico – Chemical tests: Ash value, Acid insoluble ash,Water insoluble ash, Loss on drying,Loss onignition, and pH value are some of the physicochemical tests, employed in the presentstudy. The brief review of the same is made herewith as under.1) Determination of Ash value140, 141:Definition of Ash: The residue remaining of incineration is the ash content of the drug,which represents the inorganic salts naturally occurring in drug or adhering to it ordeliberately added to it as a form of adulteration.Method: Total ash is designed to measure the total amount of material produced aftercomplete incineration of the ground drug at as low temperature as possible (about 4500)to remove all the carbons. 2 to 3gms of the air dried crude drug has to be accuratelyweighed in the tarred Platinum or Silica dish and incinerate at a temperature notexceeding 4500 C until free from carbon. Cool and weigh. If a carbon free ash cannot beobtained exhausts the charged mass with hot water, residue to be collected on ash less 48
    • filter paper, incinerate the residue and filter paper until the ash is white or nearly so.Percentage of ash to be calculated with reference to the air-dried drug.Applied aspect: Controlled incineration of crude, results in an ash residue consisting of inorganicmaterial. Total ash value represents the inorganic salts naturally occurring in drug oradhering to it or deliberately added to it as a form of adulteration. This value varieswithin fairly wide limits and therefore an important parameter for the purpose ofevaluation of crude drugs. The total ash usually consists of carbonates, phosphates,silicates and silica. High ash value is indicative of contamination, substitution,adulteration or carelessness in preparing the drug2) Determination of Acid insoluble ash142, 143:Definition: Acid insoluble ash is a part of total ash insoluble in dilute hydrochloric acid.This is a test to find out adhering dirt, silica material and sand.Method: The ash obtained by the above procedure should be boiled with 25ml of dilute Hclfor 5 minutes, the obtained insoluble matter is to be collected on Whatman’s filter paperno. 42 and washed with hot water. The residue to be taken in a crucible, dried and ignitedallowed to cool in a desiccator and weighed. The percentage of acid insoluble ash iscalculated with reference to the air dried drug.144Applied aspect: This ash value is used particularly to determine adhering dirt, silica material andsand.1453) Determination of Loss on dryingDefinition: This is a test to find out loss of moisture content in a given sample on drying.The remnant material indicates the weight of solid active substance of the given sample.Procedure146: The weight of glass–stoppered, shallow weighing bottle that has been dried underthe same conditions to be employed in the determination should be noted. Two grams ofaccurately weighed sample should be transferred to the bottle. The bottle should becovered and weight of the bottle and the contents noted. The sample is to be distributedevenly as practicable by gentle sidewise shaking to a depth not exceeding 10mm. Theloaded bottle is to be placed in the drying chamber (oven) at 1100C. 49
    • The stopper should be removed in the chamber; sample should be dried toconstant weight. After drying is completed, the drying chamber is to be opened and thebottle closed promptly and allowed to cool to room temperature. The percentage of losson drying is to be calculated.Applied aspect: It determines the amount of volatile matter (i.e. water drying of from the drug).The moisture content of a drug should be minimized in order to prevent decompositioneither due to chemical change or due to microbial contamination 147.4) Determination of pH value:Definition: pH value of an aqueous liquid may be defined as the common logarithm ofthe reciprocal of the hydrogen ion concentration expressed in grammes.148Method: One percent sample solution is to be prepared by adding 1 gm of sample mixed in100 ml water and the pH reading to be taken by using a digital pH meterApplied aspect: - pH value of a drug signifies the acidic and basic nature of the drug. This helps indetermining the pharmacokinetic property of the drug.5) Estimation of Arsenic:Procedure:Arsenic Trioxide stock Solution: Dissolve 132 mg of Arsenic Trioxide previously driesat 105 0c for 1 hour accurately weighed in 5 ml of sodium Hydroxide solution in a 1000ml of volumetric flask. Neutralize the solution with 2 N Sulphuric acid, add 10 ml moreof 2 N Sulphuric acid then add recently boiled and cooled water to volume and mix.Standard Arsenic solution: Transfer 10 ml of Arsenic trioxide stock solution to a 1000ml volumetric flask. Add 10 ml of 2 N Sulphuric acid, and then add water to make upvolume and mix. Each ml of standard Arsenic solution contains equivalent of 1 Mg ofArsenic.Test Preparation: Add a known quantity of the sample in a muffle furnace at atemperature not exceeding 4500C.To this add 25 ml of dilute Hydrochloric acid and boilfor 5 min. filter and make up to 50 ml. 50
    • ATOMIC ABSORPTION SPECTROSCOPY 149 :Principle: The principle used in atomic absorption spectroscopy was discovered in 1802by Wollaston when he observed the "Fraunhoffer lines" or absorption lines in thespectrum of the sun, yet this principle was only applied in 1955 by an Australianphysicist, Alan Walsh. The principle states that "Matter absorbs light at the samewavelength at which it emits light". Basically this means that atoms in the ground stateabsorb the same radiation as they emit in the excited state. An atom in the ground statewill absorb an amount of energy equal to the energy difference between the energy levelof the electron in the excited state and the energy level that the electron occupies in theexcited state. In Atomic Absorption Spectroscopy, the sample solution is first vaporized andatomized in a flame, transforming it to unexcited ground state atoms, which absorb lightat specific wavelengths. A light beam from a lamp whose cathode is made of the elementin question is passed through the flame. Radiation is absorbed, transforming the groundstate atoms to an excited state. The amount of radiation absorbed depends on the amountof the sample element present. Absorption at a selected wavelength is measured by thechange in light intensity striking the detector and is directly related to the amount of theelement in the sample.Process: An unknown sample in a solution is dissolved and sprayed finely, in thepresence of suitable conditions, into the flame burner of the atomic absorptionspectrometer. A cathode lamp will emit light to reach these electrons. The lamp mustcontain a cathode of the same element within the sample. This is because of the energyrequired to excite the similar electrons in the sample, hence enabling concentration to bedetermined. Most spectrometers contain a number of different cathode lamps suitable forvarious solution samples. In the lamp, taking in energy excites electrons. They jump to higher energy levelsby taking in a fixed quantum amount of energy. As they fall back down, they emit a fixedamount of light. This light radiates to the ground atoms in the sample solution, underspecific conditions. These unexcited electrons absorb the light. It is of a fixedwavelength. As the amount required to excite the electrons in the atoms is fixed,according to the radiated light, the spectrometer can detect the measure of light absorbed.In this way, the concentration of the elements can be calculated, as it is directlyproportional to the amount of element present. This is calculated in parts per million(ppm). The element is detected by an atomic absorption spectrum, by the light intensity 51
    • emitted by the sample. This is a series of coloured lines on a dark background, dependingon the element, at differing wavelengths.Applications: This process is employed in both qualitative and quantitative use. AAS is arapid method for the former, if only a few elements are being tested. However if manyelements are of interest the process can be too time consuming and uneconomical. Theusual quantitative method brackets the samples absorption spectrum with that of standardconcentrations to produce a linear calibration curve.Examples of the applications of AAS include: • Analysis of water for metals like lead, mercury and cadmium • Drug testing • Identification of unknown compositions • Analysis of rocks on space missionsX-RAY DIFFRACTION150, 151Introduction: X-ray diffraction is a novel and a powerful technique in the elucidation of 3-dimensional structure, composition of any matter. Structural features can be determinedat molecular and atomic level. It is an important technique for establishing the batch-to-batch reproducibility of a crystalline form in X-ray Powder diffraction. Randomorientation of a crystal lattice in a powder sample causes the x-rays to scatter in areproducible pattern of peak intensifies at distinct angles (θ) relative to the incident beam.Each diffraction pattern is characteristic of a specific crystalline lattice for a givencompound. Hence X-RD has become invariably indispensable tool in the pharmaceuticalindustry in the crackdown of structure.Discovery: In 1912 Walter Fredrich and C M Paul discovered that if a beam of X-Raypasses through a crystal or any matter turned in various directions.Principle: The main principle behind X-RD is the process of scattering of X-rays when itpasses through a matter in crystalline or powder state. The reason for this diffractionpattern is, X-rays are scattered in various directions by the electrons that form the outerpart of each atom in the matter. If the scattering centers are separated by distancescomparable to the wavelength of the X-rays, then interference between the X-raysscattered from particular electron centers can occur.Technique: The technique of X-RD is subjecting the powder matter or crystal matter tothe irradiation by beam of X-Rays and making a record of 3-dimensional diffractionpattern. In all X-RD procedures the transforming of these diffraction fractions into an 52
    • image (on photographic film) are involved. For interpretation construction of image isindispensable with the help of crystallographers and also from modern computers. Thisoperational process is called ‘Fourier transformation’.Diffraction as Plane Reflections: The diffraction pattern generated from single crystalor powder is an array of sharp spots. To explain these parts W.L.Bragg suggested that themechanism of X-RD could be cast in terms of reflections from regular, parallel arrays ofplanes within a crystal. Only under special conditions would the reflected radiationsinterfere constructively and a diffracted beam be observed. The required condition is thatthe angle of incidence (θ) of the incident beam obey the following relationship. Sinθ = n λ/2d Where lambda (λ) is the wavelength of incident radiation, d is the interplanarspacing and is an integer constant. This relationship is known as Bragg’s law and theta(θ) is known as Braggs angle.Methods: Laue method : lambda variable, theta fixed Rotating crystal method : lambda fixed, theta partly variable Powder method : lambda fixed, theta variable.Experimental procedure:1. Choices of radiation – While selecting radiation, the factors to be considered are Effect of unit cell size, Effect of specific absorption, Relative exposure timings.2. Sample preparation – Most using method is cylinder technique (rolling with binder technique). Here the specimen is ground to a uniform size and placed on a glass plate. A few milligrams of the sample are then mixed with a drop of colliodion until a homogeneous paste results. The paste then scooped up on a razor blade and rolled in to a rod shaped specimen with fingers.3. Powder Camera – Consists of Sample holder, direct beam catcher, Collimator, Screw, Slider. Basically the method involves diffraction of monochromatic X-rays by a powder specimen. Each particle in the specimen is a tiny crystal oriented at random with respect to the incident beam, there is a fair chance that a certain plane will be currently oriented to reflect the incident beam. Thus every set off planes will be capable of reflection.Applications: 53
    • Qualitative and quantitative phase analysis of organic and inorganic compounds. Determination of grain alignment in polycrystalline materials. Determination of exact crystallographic orientation in monocrystalline materials. Determination of micro structural properties. Determination of ratio of amorphous and crystalline fractions Determination of precise lattice parameters. Effect of heat treatment and as a tool for characterization of drug formation.PARTICLE SIZE ANALYSIS 152: What is particle size? Particles are three-dimensional objects for which three parameters (the length,breadth and height) are required in order to provide a complete description. As such, it isnot possible to describe a particle using a single number that equates to the particle size.Most sizing techniques therefore assume that the material being measured is spherical, asa sphere is the only shape that can be described by a single number (its diameter). Thisequivalent sphere approximation is useful in that it simplifies the way particle sizedistributions are represented. However, it does mean that different sizing techniques canproduce different results when measuring non-spherical particles. Particle Size Analysis is an analytical technique by which the distribution ofsizes in a sample of particulate material is measured. Particle size analyzers range fromthe historical sieve to modern automated light scattering instruments. The mostappropriate selection for a particular application depends on a number of factorsincluding the size range of interest, nature of the sample, the information required fromthe analysis, sample throughput, and price. Modern light scattering instruments arebecoming the particle analysis method of choice in most industries due to their analysisspeed (approximately 30 seconds), wide size range, ease of use, flexibility,reproducibility. Laser diffraction is a non-destructive, non-intrusive method that can be used foreither dry or wet samples. As it derives particle size data using fundamental scientificprinciples there is no need for external calibration; well-designed instruments are easy toset up and run, and require very little maintenance. Additionally, the technique offers: Awide dynamic measuring range, Flexibility, Generation of volume-based particle sizedistributions, Rapid data acquisition, Ease of Verification. 54
    • The Mastersizer 2000-Model of the instrument. The generic benefits of thetechnique of laser diffraction have been exploited and enhanced in the design of theMastersizer 2000, which is used worldwide for the analysis of a diverse range ofparticles. The Mastersizer has a fully optimized optical design which allows particles inthe size range 0.02 - 2000 micron to be characterized effectively. A range of dispersionunits ensures optimum sample presentation, and switching between units is relativelyeasy allowing different samples to be analyzed rapidly, in close succession. Automated,standard operating procedure (SOP) driven operation delivers a consistent analysis, andminimizes training requirements, whilst flexible software allows results presentation tobe tailored to the requirements of the customer. TOXICOLOGY REVIEWDefinition: Toxicology is the scientific study of Poisons153.Toxicology (from the Greek words toxicos and logos) is the study of the adverse effectsof chemicals on living organisms. It is the study of symptoms, mechanisms, treatmentsand detection of poisoning, especially the poisoning of people.History154 Mathieu Orfila is considered to be the modern father of toxicology, having given thesubject its first formal treatment in 1813 in his Traité des poisons, also called Toxicologiegénérale. Theophrastus Phillipus Auroleus Bombastus von Hohenheim (1493 - 1541) (alsoreferred to as Paracelsus, from his belief that his studies were above or beyond the workof Celsus - the Roman physician from the first century) is also considered "the father" oftoxicology. He is credited with the classic toxicology maxim, "Alle Dinge sind Gift undnichts ist ohne Gift; allein die Dosis macht, dass ein Ding kein Gift ist." which translatesas, "All things are poison and nothing is without poison; only the dose makes a thing nota poison." This is often condensed to: "The dose makes the poison".An even earlier writeron toxicology was Ibn Wahshiya, who wrote the Book on Poisons in the 9th or 10thcentury. The history of poison stretches from before 4500 BC to the present day. Poisonshave been used for many purposes across the span of human existence, most commonlyas weapons, anti-venoms, and medicines. Poison has allowed much progress in branchesof medicine, toxicology, and technology, among other sciences. 55
    • Authors of the ancient classics were very much aware of this fact and they advocatedgreat caution while using them.In the description of Visa Vega in human beings. VisaVega in animals and birds are also described.Toxicity155 is the degree to which a substance is able to damage an exposedorganism. Toxicity can refer to the effect on a whole organism, such as a human,bacterium, or plant, as well as the effect on a substructure of the organism, such as a cell(cytotoxicity) or an organ (organotoxicity such as the liver (hepatotoxicity). Byextension, the word may be metaphorically used to describe toxic effects on larger andmore complex groups, such as the family unit or society at large. Toxicity is a function ofsolubility. Insoluble compounds as well as the metallic forms often exhibit negligibletoxicity. In some cases, organometallic forms, such as dimethyl mercury and tetraethyllead, can be extremely toxic. In other cases, organometallic derivatives are less toxic suchas cobaltocenium cation.WHY TOXICITY STUDIES? o To eliminate any frankly poisonous substances from use in clinical trials. o To determine tolerable dosage range permitting an estimate of the probable tolerated dose in man. o To determine theraupeutic index which will indicate to some extent the degree to which clinical dose schedule may be varied. o To give some guidance to clinician who will be conducting clinical trials altering to potential untoward reactions. o To obtain some clue about mechanism of action of drugs under trial.Types of toxicity155 There are generally three types of toxic entities; chemical, biological, and physical. • Chemicals include inorganic substances such as lead, hydrofluoric acid, and chlorine gas, organic compounds such as methyl alcohol, most medications, and poisons from living things. • Biological toxic entities include those bacteria and viruses that are able to induce disease in living organisms.Relationship between dose and toxicity156 Toxicology is the study of the relationship between dose and its effects on theexposed organism. The chief criterion regarding the toxicity of a chemical is the dose, i.e.the amount of exposure to the substance. Paracelsus, who lived in the 16th century, wasthe first person to explain the dose-response relationship of toxic substances. 56
    • Factors influencing toxicity156Toxicity of a substance can be affected by many different factors, such as the: Pathway of administration (whether the toxin is applied to the skin, ingested, inhaled, injected), The time of exposure (a brief encounter or long term), The number of exposures (a single dose or multiple doses over time), The physical form of the toxin (solid, liquid, gas), The genetic makeup of an individual, an individuals overall health, and many others.Toxic metals157 are metals that form poisonous soluble compounds and have nobiological role, i.e. are not essential minerals, or are in the wrong form. Often heavymetals are thought as synonymous, but lighter metals also have toxicity, as exemplifiedby beryllium, and not all heavy metals are particularly toxic and some are even essential(such as iron). The definition may also include trace elements when considered inabnormally high, toxic doses. A difference is that there is no beneficial dose for a toxicmetal with no biological role. Toxic metals sometimes imitate the action of an essential element in the body,interfering with the metabolic process to cause illness. Many metals, particularly heavymetals are toxic, but some heavy metals are essential, have a low toxicity, and bismuth iseven non-toxic. Most often the definition includes at least cadmium, lead, mercury andthe radioactive metals. Metalloids (arsenic, polonium) may be included in the definition..Toxic capacity157 can mean the toxicity of a substance, possibly in relation to aspecific organism and toxic capacity can mean the capacity of an organism, organicsystem or ecosystem to contain a toxic substance or a selection of toxic substances (acompound) without showing signs of poisoning or dying.A reference dose158 :An estimate, with uncertainty spanning perhaps an order ofmagnitude, of a daily oral exposure to the human population (including sensitivesubgroups) that is likely to be without an appreciable risk of deleterious effects during alifetime. RfDs are usually derived from animal studies. Animals (typically rats) are dosedwith varying amounts of the substance in question, and the largest dose at which noeffects are observed is identified.Median lethal dose159, LD50 or LCt50 (Lethal Concentration & Time) of a toxicsubstance is the dose required to kill half the members of a tested population. LD50figures are frequently used as a general indicator of a substances acute toxicity. The test 57
    • was created by J.W. Trevan in 1927. It is being phased out in some jurisdictions in favorof tests such as the Fixed Dose Procedure., however the concept, and calculation of themedian lethal dose for comparison purposes, continues in wide use. As a measure oftoxicity, LD50 is somewhat unreliable and results may vary greatly between testingfacilities due to factors such as the genetic characteristics of the sample population,environmental factors and mode of administration.Dose-Escalation/Dose-ranging:- A trial in which the amount of the drug is eitherperiodically increased or increased with each new trial arm that is added. Used todetermine how a drug is tolerated animals or humans.Regulatory authorities : The toxicology studies has its international standardsmaintained by WHO that has the protocol from OECD.OECD GUIDELINES 160:The Organisation for Economic Co-operation and Development (OECD) (in French:Organisation de coopération et de développement économiques, OCDE) is aninternational organisation of thirty countries that accept the principles of representativedemocracy and free-market economy. The OECD is responsible for the OECDGuidelines for the Testing of Chemicals, a continually-updated document which is adefacto standard (i.e., soft law).Acute Oral Toxicity161 : The more traditional test for acute oral toxicity is the oral LD50 and one test protocol for this is found in OECD Test Guideline 401.Subacute oral toxicity161: The OECD test guidiline for Sub-acute or short term toxicitystudy is under OECD test guideline 407. 58
    • EXPERIMENTAL REVIEW Animal testing162 or animal research is the use of non-human animals in scientificexperimentation."Tatra Chaturvidha Bhutagramah,Sansvedaja, Jarayuja, Andaja Audbhija Sangnah|Tatra Purushah Pradhanam, Tasyopakaranam Anyat" !!.163 Man occupies a supreme position among all the living creatures. Hence forexperimental trial, other animals should be utilized as experimental models. Whiledescribing Anna RaksaVidhi, the effects of food contaminated with poison on variousanimals and birds are described164. These instances make it clear that, animalexperimentation was undertaken in ancient time also, though antediluvian communicationtechniques were different from those of the present day. In the present study, it is an attempt in determining the toxicity of Shuddha Mallaunder the heading of experimental study. Before toxicological studies can begin,experimental review of animal species used, study types, group size, dose selection androute of administration are the general issues studied wide infra.Species selection165: The choice of species for use in toxicological studies is acompromise between what is required or desired, and what is practical.Naturally whenperforming any toxicity study it would be ideal to use a species with the samemetabolism, pharmacokinetics and target organ susceptibility as man.The Rats-Animals used in the present study:166 The most widely used strains of rat are Sprague-Dawley and Wistar,the latter is aslightly smaller animal which may offer an advantage for some studies.The animals mustbe well examined which is free not only from common diseases but also free from allpathogenic organisms.Such SPF animals are usually Caesarian derived and thenmaintained under special barrier conditions.To minimise the biological variation it isusual ,when conducting toxicological studies ,to use genetically homogenous SPFanimals.On arrival in the facility167:The animals should be marked ,for identificationpurposes,and allowed to acclimatise to their new environments and diet.During this timevarious pretest measurements can be made. 59
    • 167Age Of Rats : It is desirable to commence toxicology studies in young rodents,six orseven weeks old,the animals should be about six weeks old and fully weaned on arrival.Recording Weight 167: The rats should be weighed on several occasions before the studybegins,and those failing to gain weight at a normal rateor outside a normal weight rangefor their age should be rejected.Steps to be followed before starting the experiment167: Adequate number of animals should be available for study. Each animal must be individually tested. Injuries,abnormalities and lesions should be rejected. Before allocated to groups rats must undergo basic haematological and biochemistry tests. Record the weight of all animals.Behaviour167 : Rats are usually friendly and amenable animals if handled gently,although there are some strain differences. Rats have a tendency to be nocturnal. Feeding,drinking and mating all usually occur at night. Their eyesight is poor, and blind rats willbehave as if perfectly normal.Housing167 : Rats may be kept in metal or plastic cages. If mesh floors are used, caremust be taken to ensure that the mesh is small enough that young animals do not fallthrough it. Solid bottomed cages are best and wood shavings, wood chips or paper maybe used as bedding. Cages should be clearly marked, with an individual label for eachanimal. Cages should be arranged on racks in such a way as to distribute animals fromdifferent groups evenly in the various positions in the rack and around the animal room.All these steps can overcome the ‘animal placement effect’.Standard diet 167: Rats, like all rodents, are coprophagic. They can be fed adlibitum forrestricted time on a complete pelleted rodent diet, from hoppers suspended above thefloor of the cage. The diet should contain 20-27% protein. Rats will eat 5 g feed per 100 gbody weight daily.Water167 : Water may be provided by sipper tubesor by automated watering systems. Thewater may need to be acidified or chlorinated to reduce contamination. Particularly forimmunocompromised rats. Rats will drink 5ml water per 100 g body weight daily.Environment 167: Rats are less sensitive to temperature changes than mice, but should bekept between 21±20 C. The humidity should be 40-70%. A 12- hour light period isadequate for rats, but, being nocturnal, bright light is deleterious particularly for albinorats, and results in retinal degeneration. The level should be less than 400 IX or 100 IX 60
    • for albinos. Photoperiod affects the oestrus cycle, and 12-16h light is best for optimalbreeding.Housing Environment has been observed to influence the toxicity of somedrugs.Breeding167 : Puberty occurs at 50-60 days, and breeding begins at 3 months whenfemale weigh 250 g and makes 300 g. They breed until they are 12-18 months old.Oestrus occurs every 4-5 days. Mating usually occurs at night, and a copulatory plug ofgelatinous material is left in the vagina of 12-24 hours, which then falls out and can bedetected to confirm that mating has occurred. Gestation lasts 21-23 days. A litter of 6-12pups is born in a shallow nest made by the female.Growth : Male rats exhibit prolonged growth, and bones do not become fully ossifieduntil their second year.168Group Sizes169: The minimum number of animals in treatment groups is dictated by theregulatory authorities.It may be advisableto to increase the numbers of experimentalanimals as there is a desire of accuracy and sensitivity.Normally 6-10 rats in a group isappropriate.Route of administration170: As a general rule drug administration in animals must include the route to beused in human dosing ,as well,usually,as the oral route.The method of dosing aredependent upon the circumstances of the study.When rodents are used the drug is usuallygiven as a suspension or solution by gavage.Dosing by gavage also has its problems.Itrequires skill on tha part to avoid dosing accidents.high doses of hypertonic ,irritants cancause toxicity in the respiratory tract by reflux from the oesophagus to the trachea.Suchproblems can be minimised by using suitable catheters.Dose selection171: The usual way of conducting the toxicological study,as advocated by theregulatory agencies, is to use three dose high,medium and low,design with a negativeplacebo or vehicle dosed control group.The basic idea is to assess the target organtoxicity directly and and to determine a threshold effect.By charecterising the toxicresponse over an experimental dose range it is possible not only to compare the relativetoxicity of the test agent but also to determine a NOEL (no observed effectlevel).Determination of NOEL is important in order to ensure that the dose of drug whichproduces the desired pharmacological response is lower than that responsible for causingany toxicological side effects. 61
    • In acute,Subacute,and Subchronic studies ,when attempting to identify targetorgans of toxicity ,the dose may be so high as to cause death.A dose that causes a 10%reduction in body weight gains one that causes minimum toxicity.It is usual to include atleast one intermediate dose ranging group,which can provide information on dose-relatedeffects.An important point about dose selection is that,while animals may receive similardoses in terms of mg/kg body weight ,pharmacokinetic analysis may demonstrate markeddifferences in terms of drug blood concentration and or drug retention time. Study Protocol : The present study is carried on two main protocols, design for Acute and Sub-acute models which are described as following: Acute toxicity172 describes the adverse effects of a substance which result eitherfrom a single exposure or from multiple exposures in a short space of time . To bedescribed as acute toxicity, the adverse effects should occur within 14 days of theadministration of the substance. It is obviously unethical to test for acute (or chronic) toxicity in humans. However,some information can be gained from investigating accidental human exposures (e.g.factory accidents). Otherwise, most acute toxicity data comes from animal testing or,more recently, in vitro testing methods and inference from data on similar substances.Steps to be observed: To establish the maximum tolerated dose.(the highest dose not causing death or life threatening toxicity) and the minimum lethal dose. Single dose studies to establish target organ toxicity. Determination of precise LD 50 or median lethal dose.Type 1173: The group of rats are left untreated for 14 days after a single clinical dose onfirst day,during which time they are obsereved daily for the development of clinicalsymptoms.On day 15 they are killed and subjected to a thorough examination postmortem.Type 2173: To determine the target tissue during the 14 days clinical dose single clinicaldose of test agent should be administered to group of rats for 14 days and observed.It isa fixed dose procedure. On day 15 they are killed and subjected to a thoroughexamination post mortem.Type 3173: To establish a precise LD 50 the purpose of dose ranging study is fulfilled.Toinvestigate the toxicity of repeated doses of an agent at dose levels ranging from a verylow multiple of the therapeutic effective dose (somewhere in the range of ED 50 or the 62
    • anticipated human therapeutic dose range ), up to doses approaching the minimum lethaldose.The procedures used can vary but usually involve exposing experimental animals tovarious concentrations of the drug i.e from a pharmacological dose to minimum lethaldose or vice versa.The increasing dose levels in a step wise sequence until a maximumtolerated dose is reached.As the animals tolerate the treatment day by day the dosewill beraised until the highest dose tolerance is achieved.This is called an ascending phasemaximum tolerated dose (MTD) study.The dead animals are dissected for thehistopathological studies.In the present study the drug dosage was for single therapeutic dose for one day,singletherapeutic dose for 14 days,dose ranging for 14 days.Thus such a method would provideinformation on limiting toxicity and assist in establishing the maximum dose to be usedin subsequent subacute studies.In the rodents three types of toxicity study may beperformed.All the data obtained from the above studies provide further information on the toxic andlethal potential of the drug and assist in dose selection for subsequent toxicity studies.Subacute Toxicity Study174: Also meant as short term toxicity study conducted to screenfor potential adverse effects using rats as surrogates.This study may be of varyingduration generally 1 to 4 weeks or 28 days which has two purposes: To observe the possible toxicity for 28 days after administration of a single therapeutic dose on first day. To observe the possible toxicity for 28 days with a single therapeutic dose administered daily.The purpose of these studies is an investigation to identify the target organs or systems atrisk .It also gives an account of predictive adverse effects and identify NOAEL..Twoanimals must be sacrificed from all groups to evidence such changes.MEASUREMENTS AND OBSERVATIONS175 : Assessment of the behaviour of animals should be carried out informallyon a daily basis before dosing and throughout the study.Rodents should be placed on aflat surface and their posture ,movements and behaviour observed.They should then bechecked for condition of the fur and for the damaged areas of skin,subcutaneousswellings or lumps being measured ,abdominal distension etc.,All natural orifices shouldbe checked for discharges,blockages,,blood,mucus,etc.,the eyes examined fordullness,dryness,discharges,opacities,pupil diameter,ptosis,exopthalmous etc.,the colourand consistency of the faeces should be noted and any wetness or soiling of the perinium 63
    • is recorded.The mouth should be examined for excessive salivation,lumps,cuts etc.Anybreathing abnormalities such as sneezing ,wheezing,rattles of dyspnoea must be recorded.Body weight176: Changes in the body weight can be a sensitive and important monitor ofhealth of an animal.Loss of body weight,failure to gain body weight at a normal rate,isfrequently the first indication of the onset of an adverse effect.LABORATORY PARAMETERS177: To provide a valuable clues and indications of the pathological changesoccuring as a result of drug treatment we need to use similar measurements.We are notinvolved in making a clinical diagnosis but rather attempting to detect the treatmentrelated effects,untreated control groups are almost always included in toxicity studies toprovide greater precision in identifying toxicological responses.Clinical chemistry andhaematological measurements can provide can provide useful information on earlytoxicological effects,target organ toxicity and reversibility of effects,etc.Blood Sampling techniques178: For rodents a number of methods can be used.Verylarge samples can be taken under general anaesthesia from the venecava,but thisprocedure is one from which the animals are not permitted to recover.Retro-orbitalpuncture are the widely used methods to obtain the samples.Volumes of upto 2ml can beobtained from rats.In general blood samples are collected at the beginning during and atthe end of treatment.Haematological measurements179: This is to identify the the toxic effects on cellular constituents of blood .Toassess the effects on red cells it is usual to perform RBC,Hb,PCV etc.,Such parametersprovide information on the estimation of the number of circulating red cells,the oxygen –carrying capacity of the blood and the volume of red cells expressed as a fraction of totalblood volume respectively.Blood Chemistry180: It is of great importance since they provide early indications of atoxic dose;assist in identifying target organs toxicity and provide the information ofreversibilty of such effects.TERMINAL STUDIES181: At the end of the treatment,usually 24 hours after the last dose,theexperimental animals are given a final thorough examination for any externalabnormalities and then killed.Rats are usually deeply anaesthetised by exposure to overdose of diethylether vapour.When it is certain that they are deeply anaesthetized andinsensitive to pain,i.e cessation of the palpebral reflex,they are usually exsanguinated.Thestandard selection of organs and tissues are removed from the body cavity and stored in a 64
    • preserving fluid (fixative) for later histopathological examination.Changes inhistopathological responses if present then changes in organ weights become ofsecondary importance.GUM ACACIA- Vehicle drug and as control.182 This is the dried gummy exudates obtained from the stems and branches ofAcacia Senegal or other African species of Acacia. It has no pharmacological action andinert. This is used as a suspending agent for the oral administration of the trial drug andas a control drug in 1% strength..Controls: A concurrent control group which is identical in every aspect to the treatedgroups,except for exposure to the test substance,should be used.Statistical Methods183, 184,185: Descriptive statistical analysis has been carried out in the present study.Results on continuous measurements are presented on Mean ± SD (Min-Max) and resultson categorical measurements are presented in Number (%). Significance is assessed at 5% level of significance. Analysis of variance has been used to find the significance ofstudy parameters between the groups, Repeated measures ANOVA has been used to findthe significant changes of parameters with in each group. Percentage of change frombaseline has been computed in the present study to study the pattern changes of toxicityduring the study period.Analysis of Variance: F test for K Population meansObjective: To test the hypothesis that K samples from K Populations with the same mean.Limitations: It is assumed that populations are normally distributed and have equalvariance. It is also assumed that samples are independent of each other.Method. Let the jth sample contain nj elements(j=1,2,…K). Then the total number ofelements is xijN = ∑ nj x. j = ∑ nj n1 n1 ∑ ∑ ( x1 − x. j) i −1 2 ∑ nj( x. j − x..) i −1 2 S1 = S2 = 2 2 N−K K −1F=S22/S12 Which follows F distribution (K-1, N-K) 65
    • PHARMACEUTICAL STUDY This section deals with the shodhana of Malla by Swedana Procedure.Aims and objectives: The aim of present study was intended to carry out Shodhana of Malla by asingle Shodhana procedure in Post Graduate pharmacy section of Taranath GovtAyurvedic Medical College, Bellary.The objectives include: • Selection of raw materials. • Shodhana of Malla by Swedana procedure. The materials and methods were selected according to principles of Rasashastraand practical experience.Materials: • Major raw material. • Other raw materials. • Yantras and Upayantras.Major raw material:The major raw material of the present study is Malla. 500gms of genuine Malla wascollected from the local market fulfilling the Grahya Lakshanas explained in classics.Other raw materials: The other raw materials used for the present study are Karavellaka swarasa(Momordica Charantia), Water, etc.Yantras: Dolayantra and Khalvayantra were used in the present study.Upayantras: Steel vessels, mud pots, weighing machine, Gas stove, Wooden rods, knife,mixer, Thread, Kora cloth, pH paper, Match sticks, Spoons etc.Method: The whole method includes : Shodhana of Malla by Dola Yantra Swedana procedure.Methods selected for shodhana in the present study: • Swedana in Karavellaka swarasa (bitter gourd juice) for 2 yamas (6hrs). The shodhana was done according to classics, the procedures and observations are explained under the heading of experiments. 66
    • EXPERIMENT :1.Shodhana of MallaDate of commencement : 06.09.2007Date of completion : 06.09.2007Reference : R T 11/136.Materials : Malla: 1–50gms in 1st pot. Malla: 2-50gms in 2nd pot. Karavellaka Swarasa 3450 ml to Malla: 1 Karavellaka Swarasa 3650 ml to Malla: 2Equipments : Mud pot, gas stove, steel vessel, wooden rod, thread, Weighing machine, mixer.Principle : Swedana in Dola yantra containing Karavellaka Swarasa.Procedure: Preparatory procedure followed were two in number.A. Extraction of Karavellaka Swarasa: About 111/2 kgs of Karavellaka fruits were taken, cut at the center andremoved the seeds and juice was extracted by using mixer, filtered through a clean clothand measured, which was about 9 ltrs.B. Preparation of Dola yantra: A wide mouthed mud pot was taken, a wooden rod was placed over the rimhorizontally. Half of the vessel was filled with the Karavellaka Swarasa which was1ltr. Aclean, white, square shaped kora cloth measuring 10 inches in length and 10 inches inbreadth was taken. 50gm of small pieces of Malla were placed at the center and a pottaliwas made and strong thread was used to tie the pottali. This pottali was suspended againby tying to the wooden rod placed on the rim of vessel containing Karavellaka Swarasa.Main procedure: Properly prepared Dola yantra was kept on gas stove for heating. Mildheat (Mrudvagni) was given for 6hrs(2 yamas). Karavellaka Swarasa was added as andwhen required to maintain the minimum liquid level. After 6hrs(2 yamas), heating wasstopped and the contents were allowed to self-cooling. Then the pottali was removedfrom the liquid and washed thoroughly with hot water. Then the pottali was opened and 67
    • Malla was collected and washed thoroughly with hot water and kept spread for drying ona clean white cloth.Observations:: • Light Green colored swarasa turned to brownish yellow color at the end of the procedure. • At the end consistency of liquid mixture was thick when compared to fresh condition. • 1ltr of Karavellaka swarasa was sufficient to immerse the pottali. • For every 30-40 mins about 100-150 ml 0f swarasa was added as the level reduced. • The Karavellaka swarasa required for whole process was 4.5 ltrs. • A characteristic smell of Karavellaka was felt during initial stage of boiling. • Greenish yellow scum had started to accumulate at the end of half an hour. • The Greenish yellow scum was found over the pottali at the end of swedana. • The solution became more acidic. • Swarasa contained minute particles which tend to sediment with the time. • Before subjecting to swedana, the color of Malla was white and after subjecting to swedana also there is no change in color. • After complete drying, the Malla was white in color with slight shining. • There was a minimum change in weight of malla. • Temperature range 900C -970C. Table No 8: Showing the observation during Malla Shodhana Particulars Before Shodhana After Shodhana Colour of Karavellaka Light green Brownish Green Swarasa Increased with suspending Consistency of swarasa Liquid and Clear Particles Natural fresh odour of Odour of the Swarasa Very Obsessive odour swarasa 68
    • pH of swarasa 6.84 (Acidic) 6.72 (More acidic) Colour of Malla White White Appearance Shiny/glassy Opaque dull. 1)50gm 1)46gm Weight of Malla 2)50gm 2)47gmPrecautions: • Sufficient quantity of Karavellaka swarasa was taken to immerse the pottali completely. • The liquid in the vessel is maintained to immerse the pottali throughout the procedure. • The liquid was added according to requirement. • The pottali was tied properly to avoid spilling of small pieces of Malla (from pottali). • The pottali was maintained in such a height that it did not touch the bottom of the vessel. • Mild heat was maintained throughout the process.Results:Weight of Malla before shodhana : 1st Pot - 50gm, 2nd Pot- 50gm.Weight of Malla after shodhana : 1st Pot- 46gm, 2nd Pot- 47 gmLoss of weight : 1st Pot- 4gm., 2nd Pot- 3 gm. 69
    • ANALYTICAL STUDY Drug analysis consists in the estimation of purity and quality for drugs usedin the Pharmaceutical preparations. The drugs that are manufactured should beunderstood well and vividly interpreted in the light of modern chemistry. A qualitycontrol program for Pharmaceutical industries involves, batch to batch uniformity of theproducts ensuring that the final product posses desired characteristics of identity, purity,potency, uniformity, safety, efficacy and stability with in established levels which meetall the legal, professional and company standards. When these things applied toAyurvedic formulations, helps in standardizing the drug in total, for better understanding& interpretation of Physico-chemical changes occurring in processing’s and also ensurethe safety and efficacy of the drug.In present study analytical study was done for the following two samples i.e. • Ashuddha Malla • Shuddha Malla.Aims and Objectives: • To analyze physicochemical properties of Ashuddha Malla and Shuddha Malla. • To carryout quantitative estimation of Arsenic (As %), in Ashuddha Malla and Shuddha Malla. • To carryout X-Ray diffraction studies on Ashuddha Malla and Shuddha Malla.Materials and methods:Materials: • Ancient parameters were conducted at P.G Dept. of Rasashastra, T.G.A.M.C., Bellary. • Modern Physical and chemical tests were conducted at Ganesh Consultancy and analytical services, Mysore. • An X-Ray diffraction study was conducted at Indian Institute of Science, Bangalore. 70
    • Methods:Physical tests:Ancient parameters: The ancient parameters such as Varna, Sparsha, Gandha were studied at PostGraduate Department Rasa Shastra, T.G.A.M.C. Bellary. Following results wereobtained.Varna : White. (Shwetha)Sparsha : Smooth, fine (mrudu).Gandha : Odorless. (nirgandha)Modern Parameters: The physical tests of two samples of Malla i.e. Ashuddha Malla and ShuddhaMalla were carried out at Ganesh Consultancy,Mysore. The tests included OrganolepticCharacters, Estimation of Total Ash, Determination of Acid insoluble Ash, percentage ofArsenic, Determination of loss on Drying at 1100C, loss on ignition and pH.Table No 9: Showing the Organoleptic Characters of two samples of Malla. DRAVYA VARNA SPARSHA GANDHA Ashuddha Malla Pure White Smooth, fine OdorlessShuddha Malla Dull White Smooth, fine OdorlessDetermination of Ash value:Materials: 1. Silica crucible. 2. Electronic weighing machine. 3. Electric furnace. 4. 2gms of two samples of Malla each i.e. Ashuddha Malla and Shuddha MallaProcedure: Two grams of accurately weighed, grounded two samples of Malla weretaken separately and transferred to the cleaned, dried and weighed Silica crucible,previously ignited and weighed. Scatter the ground dry in a fine even layer on the bottomof the crucible. Incinerate by gradually increasing the heat not exceeding dull red heat(4500C) until free from carbon. Cool and weigh. The ash value of two samples of Mallawas calculated with reference to the air-dried drug.Results:The percentages of total Ash value of two samples of Malla are as follows; 71
    • Ashuddha Malla : 8.92% Shuddha Malla : 8.86%Determination of Acid Insoluble Ash:Materials: 1. Silica crucible. 2. Burner. 3. Whatman filter paper. 4. Electronic weighing Machine. 5. Diluted Hcl – 25 ml. 6. Conical flask.Procedure: The Ash obtained by above procedure was boiled for 5 minutes with 25 mlof dilute Hcl. It was filtered through Whatman filter paper and was washed with water.The residue was dried, ignited in a crucible and was allowed to cool. Acid insoluble ashwas collected and the dry weight of acid insoluble ash was estimated with reference tothe air-dried drug.Results: The percentages of Acid Insoluble Ash value of two samples of Malla are asfollows; Ashuddha Malla : 0.77 % Shuddha Malla : 0.48 %Determination of Loss on drying at 1100C:Materials: 1. Silica crucible. 2. Electronic weighing machine. 3. Electronic air oven. 4. 2gms of three samples of Malla each i.e. Ashuddha Malla and Shuddha Malla.Procedure: Two grams of two samples of Malla each i.e. Ashuddha Malla and ShuddhaMalla were taken separately in a silica crucible. The weight (W1) of the crucible alongwith two grams of above said samples was noted. The samples were then dried in a hotair oven at 1100C till a constant weight is obtained and the dry weight (W2) wasobtained. The percentage of loss in weight on drying was estimated by difference inweights (W1-W2).Results: 72
    • The percentage of Loss on Drying at 1100 C of the two samples ofMalla are as follows;Ashuddha Malla : 0. 09%Shuddha Malla : 0.12 %Determination of loss on ignition:Weigh a silica crucible previously ignited for onehour at a temperature not exceeding 5000c. and cool in desiccators. Transfer to thecrucible accurately weighed sample of Shuddha Malla. Weigh the crucible accurately.Place the loaded crucible in the muffle furnace and ignite the crucible to 5000c, untilconstant weight is indicated. Calculate loss on ignition with reference to the air drieddrug.Result: The percentage of loss on ignition of the two samples of Malla is as follows,Ashuddha Malla : 82.84 %Shuddha Malla : 81.39 %Determination of water soluble ash:Boil the ash for five minutes with 25 ml of water;Collect the insoluble or matter in a Gooch crucible,ashless filter paper,wash with hotwater,and ignite for fifteen minutes at a temperature not exceeding 450°C .Substract theweight of the insoluble matter from the weight of the ash,the difference in weightrepresents the water soluble ash.Calculate the percentage of water soluble ash.Calculatethe percentage of water soluble ash with reference to air dried drug.Results: Water soluble ash of the two samples is as follows,Ashuddha Malla : 0.04 %Shuddha Malla : 0.05 %Determination of pH Value:Materials: 1) Digital pH meter. 2) Water – 100 ml. 3) Two samples of Malla i.e. Ashuddha Malla and Shuddha Malla:Procedure:1% of the two samples of Malla each i.e Ashuddha Malla and Shuddha Mallasolution was prepared (1 gram in 100 ml water) and the pH reading was taken using aDigital pH meter.Results: pH of the two samples of Malla i.e. Ashuddha Malla and Shuddha Malla;Ashuddha Malla : 6.98 73
    • Shuddha Malla : 7.08Qualitative and Quantitative chemical analysis: Qualitative and quantitative chemicalanalysis of the two samples of present study was taken up at Ganesh ConsultancyServices,Mysore.Estimation of Arsenic:Methodology: Instrumental method using atomic absorption spectroscopy.Materials: Atomic Absorption Spectrophotometer with a graphite tube cell.Make: Analytik Jena AAS, Germany.Model: Nov AA 4OOG.Sample: Ashuddha Malla & Shuddha Malla each 3 gms.Parameters: Wavelength - 193.7hm Drying temperature - 1100C. Thermal decomposition (Phyrolysis) - 11000C. Atomization-2,2000C Working range - 10ppb-50ppb.Method: Weigh 3gm of sample in silica crucible and ashed at 5000C in a Muffle furnace,after cooling and add 5ml of con. hydrochloric acid. Bril, cool, filter and make up thevolume to 100ml. Further dilutions done, if required to attain the working range of theinstrument.Optimise the response of the instrument for wavelength setting etc. Atomizeblank to get Zero absorption. then atomize the Standards (minimum 3 standards) andnote down the absorption. Then atomise the sample to get absorption value of thestandard against concentration in Kg/Litre of Arsenic. Locate the point of the sampleabsorption and calculation of the concentration of Arsenic in the sample.Calculation: Percentage of Arsenic: C X V X 100 109 MWhere, C: Concentration of Arsenic in Mg/litre in the final Solution V: Volume in ml of the final Solution. M: Mass in gram of the sample in the final Solution.Results: The quantitative & qualitative estimation of the Arsenic (As%) of the twosamples of Malla i.e.Ashuddha Malla and Shuddha Malla are as follows; Ashuddha Malla : 69.72% Shuddha Malla :74.22% 74
    • PARTICLE SIZE:- Laser Particle Size Analysis consists in measuring the size of particles of thesample using the diffraction and diffusion of a laser beam. During the laser diffractionmeasurement, particles are passed through a focused laser beam. These particles scatterlight at an angle that is inversely proportional to their size. The angular intensity of thescattered light is then measured by a series of photosensitive detectors. The map ofscattering intensity versus angle is the primary source of information used to calculate theparticle size. The Mie scattering model accurately predicts the scattering of particles. MieTheory allows for primary scattering from the surface of the particle, with the intensitypredicted by the refractive index difference between the particle and the dispersionmedium. It also predicts the secondary scattering caused by light refraction within theparticle . The particle size of the drugs both Shuddha Malla and Ashuddha Malla wasanalysed at Dept.of Materials engineering, IISc ,Bangalore using Model Malvern’sParticle Size analyzer 2000 for Laser diffraction method.Table 10: Showing the Summary of particle size analysis. Volume Shuddha Malla Ashuddha Volume Under % Under % Size(µm) Malla Size(µm) 0.04 0.06 0.02 0.06 1.34 0.13 1.65 0.15 10.35 0.36 11.81 0.31 48.78 41.43 48.64 22.49 94.17 258.95 94.78 76.32 100.00 477.01 100.00 120.67The d50 (50%) of particle size of Shuddha Malla is : 43.32µm.The d50 (50%) of particle size of Ashuddha Malla is : 23.67µm. 75
    • X-Ray Diffraction (XRD)XRD of all samples were carried out at Indian Institute Of Science,Bangalore.Analytical procedure of XRD: For the present study the X-ray analysis of two samples were carried out usingJEOL X-ray diffractometer. The machine is equipped with Cu K-alpha (Lambda-1.5406)radiation and graphite monochromator operated at 40 kV / 30 mA). All samples werevery well grounded to 200 meshes and air-dried. The X-ray diffractometer scans wasmade on randomly oriented samples from 3-65° 2-theta (d=29.42 to 1.43 angstrom) witha step size of 0.02° and 1 second time per step. The 2-theta value and intensity of the peak (counts) are represented on X and Y-axis respectively. Please note that higher the value of counts represents higher thecrystallanity of the phase. The crystallanity varies sample-to-sample and experimentalconditions. For identification of each phase, minimum 5 strong peaks were chosen andcompared with standard JCPDS. The standard JCPDS card number, identified phase andcomposition are also given.X-Ray diffraction study:Table No 11: Shows the chemical composition of 2 samples confirmed afteridentification of d-identified, d-standard peak values, 2 theta standard and 2 thetaidentified peak values.Sample :1 Ashuddha Malla. Sample : 2 Shuddha Malla. Sample:2Highest 4771 Standard 100% 100% D 3.195 D 3.192 27.902° 27.948° RelativeCounts Intensity Standard Identified 2 theta 2 theta 1498 28% 31% Intensity 2.7680 2.538 32.315° 35.361° Peak Peak standard identified 932 21% Sample :1 20% 1.6700 Values 2.766 Values 54.935° 32.366° 6858 856 100% 17% 100% 18% 3.195 2.1320 3.211 1.957 27.902° 42.359° 27.779° 46.403° 1885 27% 27% 1.9570 2.55 46.358° 35.198° 12% 654 1426 21% 14% 21% 2.2620 1.6700 1.551 2.78 39.818° 54.935° 59.605° 32.203°Crystal Structure of As2 O315% 997 17% 2.1320 : Face centered cubic. 1.963 46.358° 46.244° 689 10% 10% 1.3530 1.674 54.935° 54.84° 76
    • Sample :1:Ashuddha Malla • JCPDS card no:04-566 • Composition: Arsenolite,As2O3 • Scan date:28.08.2008 • Ref:Swanson,Tatge,Natl.Bur.Stand (U.S),Circ.539,1.51 (1953) • Color:ColorlessPattern at 26O C.A Mallinckrodt Sample. It was recrystallised bysublimation.Spectroscopic analysis shows Fe,Mg,Sb,Si 0.001 to 0.01% each.Ca and Pbtrace.Isostructural with senarmontite.Sb2 O3. • As2O3 type.Also called Arsenic glass,WhiteArsenic,.PSC:cF80. • Molecuar wt:197.84 • Unit Cell Volume:1358.41Foot Note 1) Ashuddha Malla : Totally 25 peaks were identified in the sample from 30-840 2-theta at x-axisand from 0-8000 counts on y-axis. (d =6.453 to 1.162 angstrom). In this 5 peaks werechosen as strong peaks with their relative intensities with standard JCPDS. 1st peak wasconsidered as significant at 27.779°, d observed was 3.211 compared to 3.195 withrelative intensity of 100%, 2nd peak was considered at 35.198°, d observed was 2.55compared to standard d 1.9570 with relative intensity of 27%. 3rd peak was considered at32.203°, d identified was 2.78 compared to 1.6700 with relative intensity of 21%. 4the 77
    • peak was considered at 46.244°, d identified was 1.963 compared to Standard 2.1320only, with relative intensity of 15%. 5th peak was considered at 54.84°, d identified was1.674 compared to 1.3530 with relative intensity of 10%.Sample 2: Shuddha Malla • JCPDS card no:07-400 • Composition:Arsenolite,As2O3 • Scan date:28.08.08 • Ref:Calculated from ICSD using POWD-12++.(1977) • Ref: Pertlik, F.,Czech, JPhyscs..28.170 (1978) • As2O3 type.PSC:cF80 • Molecular wt:197.84 • Unit Cell Volume:1358.04Foot Note 2):- Shuddha Malla Totally 25 peaks were identified in the sample from 30-840 2-theta at x-axisand from 0-8000 counts on y-axis. (d =6.453 to 1.162 angstrom). In this 5 peaks werechosen as strong peaks with their relative intensities with standard JCPDS. 1st peak wasconsidered as significant at 27.779°, d identified was 3.211 compared to 3.195 withrelative intensity of 100%, 2nd peak was considered at 35.198°, d identified was 2.55compared to standard d 1.9570 with relative intensity of 27%. 3rd peak was considered at32.203°, d identified was 2.78 compared to 1.6700 with relative intensity of 21%. 4thpeak was considered at 46.244°, d identified was 1.963 compared to Standard 2.1320only, with relative intensity of 15%. 5th peak was considered at 54.84°, d identified was1.674 compared to 1.3530 with relative intensity of 10%. 78
    • EXPERIMENTAL STUDY Knowledge of the mode of action of a drug obviously greatly enhances predictionfrom animal studies of what will happen in man. Mineralo-metallic forms that aredeveloped into drugs must have therapeutic efficacy and be safe. Therefore, in thedevelopment of drug it is essential to select drugs that have a margin of safety betweenthe dose that produces the desired (therapeutic) and the dose that produces undesired(toxic) effects. The margin of safety, for some drugs is small, and some peopleintentionally overdose themselves. As a result, toxic effects of drugs often are observed. The experimental study should be well planned and various inferences are to bedrawn with respect to therapeutic dosage, route of administration and safety. Theevidences collected out of the experimental study should be accurate and beyond the biasand errors so as to get reproducibility and hence accountability. Thus Rasadravyas andAyurvedic formulations can be brought into GMP as well as GCP. The task of animal experiments is to minimize the possibilityof missing the useful drug and to obtain maximum information from relatively feweranimals. Animal experimentation for screening of drugs has a number of limitations likeinterspecies variation still the need of the hour is Safety. The present study is one such attempt in determining the toxicityprofile, acute and sub-acute of the drug Shuddha Malla which is a potential toxicophore,simultaneously is one of best drugs described therapeutically in recommended dosesclassically as seen in review. Such Pre-clinical database is not been obtained until now.Thus its time for proving the safety related studies through toxicology.Locale of the Study:- The toxicological studies was conducted according to the WHOprotocols for screening the drug Malla (As2O3) for toxicology in the Department ofPharmacology and Toxicology, Veterinary College, Karnataka Veterinary, Animal andFisheries Sciences University, Hebbal, Bangalore -560024. 79
    • ACUTE STUDYThe initial acute study is generally a single dose and a single dose with 14 days of shortterm repeated dose study observation186. It is necessary to run a more comprehensiverange of dose levels in short term repeated dose study which increases the chances ofdefining the dose response188.The present study is for 14 days period in which Shuddha Malla is the test drug.Study Start Date: 5th October 2007Study End Date: 3rd November 2007Objective To screen the probable toxicity of the drug Shuddha Malla on albino rats with a single dose. To screen the potential toxicity of the drug Ashuddha Malla on albino rats with a single dose. To screen the probable toxicity of the drug Shuddha Malla on albino rats with repeated doses for 14 days. To screen the potential toxicity of the drug Ashuddha Malla on albino rats with repeated doses for 14 days To establish the median lethal dose of drug Shuddha Malla. To establish the median lethal dose of the drug Ashuddha Malla.Materials: Animals : Wistar Albino rats (Weighing 150-200 gms). Drug and Chemicals : • Karavellaka Shodhita Shuddha Malla. • Ashuddha Malla (raw sample before shodhana) • EDTA (Disodium ethylene diamine tetra acetic acid)(MERCK) • Diethyl ether • NBF 10% (neutral buffered formalin) (40% formalin-100ml, distilled water-900ml, Sodium phosphate monobasic-4 gm, Sodium phosphate dibasic-65gm) • Gum acacia. 80
    • Equipments : Digital animal weighing balance Milligram digital weighing machine. Centrifuging machine Serum biochemical semi auto- analyzer Sterilizing oven. Other accessories: Tuberculin syringe (1ml). 2ml and 5ml syringe. Gavage needle for intra gastric or oral administration. Surgical Set, tissue paper roll, cotton roll Storage Vials 5ml pp, Capillary tube 2mm bore Micro centrifuge tube 2ml pp Plastic containers. Surgical gloves.Methods:Pre procedure:1) Selection of animal species: Healthy Albino Rats of either sex weighing 150-200gms were randomly selectedand used in the present study. The animals were obtained from the Central animalHouse,Indian Institute of Science, Bangalore.2) Housing and feeding conditions: Animals were kept in polypropylene cages with paddy husk bedding. Sexdistribution was maintained equal in all age groups. The temperature in the experimentalroom was around 240C. Lighting was natural, the sequence being 12 hours dark, 12 hourslight. They were provided standard food pellets procured from Amruth Feeds,B’lore andwater ad libitum. The experimental procedures were carried out in accordance with the ethicalguidelines for animals proposed by the CPCSEA , Government of India. The approvalof the ethical committee, Karnataka Veterinary, Animal and Fisheries SciencesUniversity, Hebbal, Bangalore was obtained before starting the experiments. 81
    • 3) Grouping: Before grouping the inclusion and exclusion criteria was fulfilled.Inclusive criteria: 1) Adult healthy albino rats from both the genders. 2) Rat weighing 150-200gms 3) Albino rats between 90-120 days were included.Exclusive criteria : 1) Unhealthy albino rats 2) Weight below 150gms and above 200gms 3) Albino rats of below 90 days and above 120 days were excluded. The remaining animals were randomly selected, 56 animals were equally dividedinto seven groups of eight animals each. In each group the animals were marked withnumbers to permit individual identification. Each group of 8 animals was kept in separatepolypropylene cages, for one week prior to dosing to allow acclimatization to laboratoryconditions.4) Examination of the animals prior to the experiment: • Heart rate was counted as number of beats/minute by feeling the heart rate by thumb. • Respiratory rate was counted as number of inspiration and expirations/minute observing the movement of abdomen.5) Fixation and preparation of rat dose: The normal human adult dose of Shuddha Malla is 1/120-1/30 Ratti, which isequal to 1mg-4mg. This was converted into animal dose based on Paget and Barner’ssurface area ratio which works out to be 0.36mg /kg body weight.i.e. Rat dose / kg body wt. = 0.018 x Human dose x 5 = 0.018 x 1/30 Ratti x 5 = 0.018 x 4mg x 5 Therefore Rat dose of Shuddha Malla = 0.36 mg / kg body weight. 82
    • Similarly, total daily dose of Ashuddha Malla is taken as 0.36mg/kg body weight.Vehicle for Administration of Drug: As the drugs are insoluble, the gum acacia 2% solution (by mixing 2 gms of gumacacia in 100 ml of distilled water) is taken as the vehicle and the same as control.The 2% gum acacia is calculated for rat dose of 5 ml /kg body weight.Procedure:Administration of Drugs: Drug was administered through intra gastric tube using 2 ml syringe fitted with 18gauze needle made of steel provided with 5 number infant feeding tube to avoid injury tothe rats during drug administration. Prescribed dose of suspended drug was loaded insyringe and the tube was inserted into the oesophagus. After confirming that the tube wasinside the oesophagus, drug was pushed slowly to reach the gastrum.Table No. 12: Shows drugs according to groups. Group Number of Rats Drug Purpose Group I 8 2% gum acacia To serve as control Shuddha Malla To serve as test trial single dose only on group and observe Group II 8 1st day & for the single observation for 14 therapeutic drug days. effect. Shuddha Malla To serve as test Group III 8 single dose for 14 trial group and days daily. observe for the single dose daily until 14 days. Shuddha Malla in To serve as test trial 83
    • Group IV 8 escalated doses for group for evaluating 14 days. median lethal dose. Ashuddha Malla To serve as test trial single dose only on group and observe Group V 1st day & for the single 8 observation for 14 therapeutic drug days. effect. Ashuddha Malla To serve as test Group VI 8 single dose for 14 trial group and days daily. observe for the single dose daily until 14 days. Ashuddha Malla in To serve as test trial Group VII 8 escalated doses for group for evaluating 14 days. median lethal dose.Foot note:-The grouping was done randomly from both the genders to prevent bias.Table 13 :Shows the weight and administered dose accordingly in eachgroup for 14 days.Group Weight of the Rat Calculation of dose Administered doseGroup I ±175 5 x 175 0.75 ml 1000Group II ±175 0.36 x 175 x 100 ml 0.63 ml 1000 10 mgGroup III ±175 0.36 x 175 x 100 ml 0.63 ml 1000 10 mgGroup V ±175 0.36 x 175 x 100 ml 0.63 ml 1000 10 mgGroup VI ±175 0.36 x 175 x 100 ml 0.63 ml 1000 10 mg Group IV and VII are administered doses in increasing range by multiples of 5each day to the therapeutic dose173. Therefore the following table shows the doses givento these groups for 14 days schedule in mg/kg body weight which was converted to mlwith gum acacia solution. 84
    • Table 14: Shows the escalated doses of the test drug charted accordinglyto Group IV & VII. (Ref: Fixed dose Procedure) 189 Group Day Day Day Day Day Day Day Day Day Day Day Day Day Day IV & 1 2 3 4 5 6 7 8 9 10 11 12 13 14 VII Malla 0.36 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 19.8 21.6 23.4 Gum 0.63 3.1 6.3 2.1 1.4 1.8 2.1 1.2 1.4 1.6 1.8 1.9 2.1 2.0 acacia ml ml ml ml ml ml ml ml ml ml ml ml ml ml solnFoot note: As the concentration of the drug increased the volume of the vehicle decreasedbecause the gastric capacity of the rats is only 4-5 ml. Both the respective groupsreceived as per the dose schedule above. These two groups are kept under observation toknow the median lethal dose. Main Study Procedure: The animals from all groups were administered their respective drug dosage everyday as shown in the above tables. General clinical observations on feed and water consumption, posture, gait, and social behaviour. The health condition of the all the animals was recorded. All the animals were observed thrice daily for morbidity and mortality. Method: Rats were placed on the flat surface to observe the posture, movements and behaviour.Gently feel from head to tail to check the condition of the fur and damaged areas of skin, areas of tenderness. All natural orifices were checked for any discharges, blockages, blood, mucus etc., the color and consistency of the faeces.The mouth examined for excessive salivation, breathing abnormalities etc. Body weight recorded: The body weight of all the rats are recorded for every 3rd day to know the significant changes. The rats from Group I, II, III, V and VI were sacrificed on 14th day to trace out any histopathological changes. Group IV and group VII are the groups with escalated doses of Shuddha Malla and Ashuddha Malla respectively which were under observation for morbidity 85
    • and mortality. Autopsy of such dead animals was carried out and preserved the organs for histopathological study. The following table gives the account of dead and alive rats in particular doses. Table 15: Showing the day and number of rats dead in Group IV . Group Day Day Day Day Day Day Day Day Day Day Day Day Day Day IV 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Dose 0.36 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 19.8 21.6 23.4 R1 A A A A A A A A A A D - - - R2 A A A A A A A A A A D - - - R3 A A A A A A A A D - - - - - R4 A A A A A A A A A A D - - - R5 A A A A A A A A A A A A A A R6 A A A A A A A A D - - - - - R7 A A A A A A A A A A D - - - R8 A A A A A A A A A D - - - -Foot note: A –alive, D-dead, R-rat number. In Group IV maximum mortality of rats wason 11th day at dose of 18 mg which is the maximum tolerated dose. The minimumtolerated dose is 14.4 mg on 9th day. The median lethal dose is calculated on this base189.Table 16: Showing the day and number of rats dead in group VII.Group Day Day Day Day Day Day Day Day Day Day Day Day Day DayIV 1 2 3 4 5 6 7 8 9 10 11 12 13 14Dose 0.36 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 19.8 21.6 23.4R1 A A A A A A D - - - - - - -R2 A A A A A A A A D - - - - -R3 A A A A A A A A D - - - - -R4 A A A A A A A A D - - - - -R5 A A A A A A A D - - - - - -R6 A A A A A A A D - - - - - -R7 A A A A A A A A D - - - - -R8 A A A A A A A A D - - - - - 86
    • Foot note: A –alive, D-dead, R-rat number. In group VII maximum mortality of rats wasseen on 9th day i.e. at the dose of 14.4 mg which is the maximum tolerated dose. Theminimum tolerated dose is 10.8 mg on 7th day. The median lethal dose is calculated onthis base.Collection of blood samples:- Blood of approximately 4 ml volume was drawn fromthe retro-orbital plexus using a capillary tube under ether anaesthesia,after an overnightfast. Blood samples, mixed with disodium ethylene diamine tetra acetic acid (EDTA) aswere used for haematological measurements. Serum samples were separated bycentrifugation at 5000 rpm for 20 min, and then were stored at - 80°C until analysis forserum biochemical measurements. Investigations: 1. Haematology:- The following haematological parameters were determined using blood samples collected on day 0,7th and 14th day using microhaematocrit capillary tubes. 1. Haemoglobin levels(Hb) 2. Total Leucocyte Count (TC) 3. Diffrential Count.(DC) 4. Packed Cell Volume.(PCV) 5. Red Blood Corpuscles (RBC) 2. Serum biochemical parameters:- Serum biochemical parameters were estimated from the serum samples collected from the animals on Day 0,7th and 14th day using a semi automatic auto analyzer and ready to use kits (Swemed diagnostics, Bangalore).The following humoral parameters were estimated :- 1. Serum Urea. 2. Serum Creatinine 3. SGOT (Serum glutamic oxalacetate transaminase)/AST 4. SGPT (Serum glutamic pyruvate transaminase)/ALT COLLECTION OF ORGANS FOR HISTOPATHOLOGICAL STUDIES:- At the end of experimental period of 14 days, the animals from all the groups were sacrificed except group IV & VII and observed for gross lesions of internal organs. The main organs like Heart,Brain,Liver,Kidney,Lungs,Spleen and 87
    • Gastric mucosa were collected separating it from adhering tissues using saline and collected by placing on blotting paper and gently pressed to remove excess of saline.The tissues were also collected from the rats which are dead due to escalated doses on particular days post-mortem however,severly autolysed were only necropsied and inspected for obvious changes.Fixation : The tissues were excised out immediately after sacrificing, cleaned ofextraneous tissue cut into pieces of 3-5 mm thickness and transferred to 10% formalinsolution. The tissues were allowed to remain in it till they are taken up for processing.Tissue Processing : Tissues were thoroughly washed by placing them under running tap water andplaced in 70% alcohol. The tissues were subjected to dehydration, clearing and paraffininfiltration by passing them through 80, 90 and 95% alcohol (2 changes), isopropylalcohol, acetone (2 changes) chloroform (3 changes) paraffin (2 changes) (3 each). Nextthe tissues were embedded in paraffin to prepare tissue block in paraffin. Tissue blockswere fixed to metal object holder after trimming them to suitable size.Section Cutting: The tissue sections (5 µm) were cut with help of a Spencer type rotarymicrotome and floated in a water bath between 40-45°C. Then they were mounted onclean glass slides with a drop of Mayer’s egg albumin, dried on hot plate at about 50°Cfor 30 min.Staining Procedure (Haematoxylin Eosin Stain) : The sections were stained by serially placing them in xylol, acetone, 95%alcohol, running water, haematoxylin stain, running water again, eosin solution 95%.alcohol (3 changes), acetone (2 changes), xylol - 2 changes and mounted with D.P.X.The slides were viewed under a microscope at various magnifications to note down themicroscopic features.STATISTICAL ANALYSIS:- The data generated from the experimental study wassubjected to one- way ANOVA (Analysis of Variance) by statistical analysis. 88
    • SUB-ACUTE STUDY Repeated dose toxicity study are conducted to screen for the potential adverseeffects of the drug chemical using lab animals as surrogates for a target species, mostoften the human. The study duration is for about 4 weeks187.A 28 day study can producemore valuable information than a 14 day study because of the long term exposureincreases the probability of detecting more slowly developing adverse effects, assumingthe dose range tested is similar188.The present study is for 28 days period in which Shuddha Malla is the test drug.Objectives: To screen the probable adverse affects of the drug Shuddha Malla on albino rats. To screen the probable adverse affects of the drug Ashuddha Malla on albino rats. Identify the target organs. Determine need for specialized endpoints to be assessed in long-term studies.Materials: Animals : Wistar Albino rats (Weighing 150-200 gms). Drug and Chemicals , Equipments , Other accessories: Same as acute study.Methods:Pre procedure: Selection of animal species, housing and feeding conditions, Inclusiveexclusive criteria and Examination of the animals prior to the experiment are all similarto Acute Studies.Grouping: The animals were randomly selected, 40 animals were equally dividedinto five groups of eight animals each. In each group the animals were marked withnumbers to permit individual identification. Each group of 8 animals was kept in separatepolypropylene cages, for one week prior to dosing to allow acclimatization to laboratoryconditions.Fixation and preparation of rat dose: The normal human adult dose of Shuddha Mallais 1/120-1/30 Ratti, which is equal to 1mg-4mg. This was converted into animal dosebased on Paget and Barner’s surface area ratio which works out to be 0.36mg /kg bodyweight.Vehicle for Administration of Drug:As the drugs are insoluble, the gum acacia 2%solution (by mixing 2 gms of gum acacia in 100 ml of distilled water) is taken as thevehicle and the same as control. The 2% gum acacia is calculated for rat dose of 5 ml/kg body weight. 89
    • Procedure: The grouping was done randomly to administer drugs accordingly.Table No. 17: Shows drugs according to groups. Group Number of Rats Drug Purpose Group I 8 2% gum acacia soln. To serve as control Ashuddha Malla single dose only on 1st To serve as test trial Group II 8 day and 14 days group. observation. Ashuddha Malla To serve as test trial Group III 8 single dose for all 14 group. days. Shuddha Malla single To serve as test trial Group IV 8 dose only on 1st day group. 14 days observation. Shuddha Malla single To serve as test trial Group V 8 dose for all 14 days. group.Table 18 : Shows the weight and administered dose for 28 days.Group Weight of the Rat Calculation of dose Administered doseGroup I ±175 5 x 175 0.75 ml 1000Group II ±175 0.36 x 175 x 100 ml 0.63 ml 1000 10 mgGroup III ±175 0.36 x 175 x 100 ml 0.63 ml 1000 10 mgGroup IV ±175 0.36 x 175 x 100 ml 0.63 ml 1000 10 mgGroup V ±175 0.36 x 175 x 100 ml 0.63 ml 1000 10 mgAdministration of Drugs: Drug was administered through intra gastric tube using 2 mlsyringe fitted with 18 gauze needle made of steel provided with 5 number infant feedingtube to avoid injury to the rats during drug administration. Prescribed dose of suspendeddrug was loaded in syringe and the tube was inserted into the oesophagus. Afterconfirming that the tube was inside the oesophagus, drug was pushed slowly to reach thegastrum.Main Study Procedure: 90
    • The animals from all groups were administered their respective drug dosage everyday (1-28 days) as shown in the above tables. Body weight was recorded on 0, 3rd, 6th ,9th ,12th ,15th ,18th ,21st ,24th ,27th days of all the groups to rule out any significant changes. Observations: General clinical observations were made at three times a day and considering the peak period of anticipated effect after dosing. The health condition of the all the animals was recorded. Thrice daily, all animals were observed for morbidity and mortality. Two animals selected randomly from all the groups were sacrificed on 7th, 14th ,21st and 28th day to trace out the organ toxicity in specific.Collection of blood samples:- The rats were anaesthetized with Diethyl ether and bloodwas collected from retro orbital plexus. The blood samples were collected in Disodiumethylene diamine tetra acetic acid (EDTA) vials for estimation of haematologicalparameters. Serum samples were separated by centrifugation at 5000 rpm for 20 min, andthen were stored at - 80°C until analysis for serum biochemical measurements.1. Haematology:-The following haematological parameters were determined using bloodsamples collected on day 0,7th 14th ,21st and 28th day using microhaematocrit capillarytubes. 6. Haemoglobin levels(Hb) 7. Total Leucocyte Count (TC) 8. Diffrential Count.(DC) 9. Packed Cell Volume.(PCV) 10. Red Blood Corpuscles (RBC) 2. Serum biochemical parameters:-Serum biochemical parameters were estimatedfrom the serum samples collected from the animals on Day 0,7and 14th day using a semiautomatic auto analyzer and ready to use kits (Swemed diagnostics, Bangalore).Thefollowing humoral parameters were estimated :- 5. SGOT (Serum glutamic oxalacetate transaminase)/AST 6. SGPT (Serum glutamic pyruvate transaminase)/ALT 7. Total albumin level. 8. Total Protein level. 9. Serum Creatinine. 10. Blood Urea Nitrogen. 91
    • Histopathological Study:- On 7th, 14th, 21st and 28th day two animals from all the five groups are sacrificedto know the significant target organ toxicity, after overnight fasting, the rats weresacrificed under ether anesthesia. Gross lesions if any were observed. The main organs like Heart,Brain,Liver,Kidney,Lungs,Spleen and Gastric mucosawere cleared off from the adnexal tissues using normal saline and placed on a blottingpaper and gently pressed to remove excess saline adhering to the organ. Therepresentative samples from the organs like liver and kidney were collected in 10%neutral buffered formalin (NBF) for histopathological study. The fixed samples were then done for tissue processing,cutting sections andstaining it as described in the previous section. The slides were viewed under amicroscope at various magnifications to note down the microscopic features.STATISTICAL ANALYSIS: - The data generated from the experimental study was subjected to one- way ANOVA by statistical analysis. 92
    • OBSERVATIONS AND RESULTSPharmaceutical :- 50 gm of Malla was taken in each of 2 different pots. After Shodhana (Swedana) of 50gm of Malla in Karavellaka Swarasa, 46gm and 47 gm of Shodhita Malla was obtained in 1st and 2nd pot respectively.Analytical:-♦ Physico-chemical Analysis ,Qualitative and Quantitative results:- Table 19 :Shows the Physico-chemical results of Samples Physical tests Ashuddha Malla Shuddha Malla PH Value 6.98 7.08 Ash Value 8.92 % 8.86 % Acid insoluble ash 0.77 % 0.48 % Water soluble ash 0.04 % 0.05 % Loss on drying 0.09 % 0.12 % Loss on ignition 82.84 % 81.39 % Table No.20: Shows Arsenic (As%) of 2 Samples of Malla. Quantitative Ashuddha Malla Shuddha Malla Analysis As% 69.72% 74.22% ♦X – ray diffraction study of 2 samples of Malla revealed that the composition of both the samples of Malla has As2O3 (Arsenolite) after comparing the d-Standard peak values in two different standards of JCPDS. ♦The Particle Size analysis by Laser Diffraction technique shows the size of Ashuddha Malla as 23.67µm and Shuddha Malla as 43.32µm. . 93
    • Experimental Results : ACUTE TOXICITY STUDY STUDY DESIGN:-There are 7 groups each with 8 animals. Group 1:Control (gum acacia 2 % solution) Group 2:Single dose of Shuddha Malla only on 1st day and 14 days observation. Group 3:Single dose of Shuddha Malla on all 14 days . Group 4:Dose escalation from 1st day -14th day Group 5:Single dose of Ashuddha Malla only on 1st day and 14 days observation. Group 6:Single dose of Ashuddha Malla on all 14 days. Group 7:Dose escalation from 1st day- 14th day. OBSERVATIONS: Behavioural Profile:- Group IV and VII are very alert compared to others initially but in later days are very passive and stereotype.Group III and VI show improved alertness as days pass. The rats from Group IV and VII are restless during initial days. Immediate touch response is observed with a spontaneous activity in Groups IV and VII in the initial days.The same sort of expression is seen in groups III and VI after 9 days. Neurological Profile:- Convulsions were seen in rats by the 6th-7th hour of high dose administration as per escalated doses which would die shortly or have an impending death. Staggering gait was seen in rats of Group IV and VII in later days. There seemed to be some resistance to Paw extension after 3-4 hrs of dosing in Group IV and VII. The Pinna reflex was normal. Autonomic Profile:- Watery Salivation seen in Group IV and VII by 8th and 6th days respectively. Diarrhoea was seen with a fluid discharge from the orifice continuously in group IV and VII by 10th and 8th days respectively. General Signs:- Writhing was noted by most of rats after a particular dosage in escalation from group IV and VII. Reduced food intake in group IV and VII around 7th to 8th day. 94
    • Retreat to the corner of the cage or excessive struggling or vocalising on dosing is seen in group IV and VII as days pass on. RESULTS OF BODY WEIGHT :- Table 21: Comparison of body weight between groups. Group I Group II Group III Group IV Group V Group VI Group VIIB.wei P valueght Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD0th 186.38 9.80 183.25 8.50 171.63 7.82 161.63 3.25 161.88 2.36 154.13 3.04 151.25 5.42 <0.001*day3rd 186.63 9.96 179.88 9.55 171.50 7.67 164.88 5.44 167.38 9.93 156.00 4.63 150.00 7.60 <0.001*day6th 193.00 12.94 186.13 9.89 178.00 9.70 168.75 5.20 168.00 5.55 158.50 5.50 151.50 9.72 <0.01*day9th 194.75 12.89 189.13 10.36 180.75 13.61 167.13 6.66 171.25 7.27 165.38 4.31 142.25 14.84 <0.001*day12t h 198.00 12.42 188.38 10.18 188.13 13.68 168.40 4.77 168.88 6.38 161.88 5.72 - - <0.01*day14t h 195.88 11.63 190.50 11.67 194.38 12.97 169.00 .- 177.50 6.91 167.88 6.27 - - <0.001*day% Change from 0th dayday +0.13 -1.84 -0.07 -2.01 -3.40 -1.22 -0.83 -3day +3.55 +2.57 -3.71 -4.41 +3.78 -2.84 +0.17 -6day +4.49 +3.21 +5.32 -3.40 +5.79 -7.30 -5.959day +6.24 +2.80 +9.61 +4.19 +4.32 +5.03 -12day +7.10 +3.96 +13.26 +4.56 +9.65 +8.92 -14 Table 22:Shows the inter group differences and its significance. P value Gr.II Gr.III Gr.IV Gr.V Gr.VI Gr.VII Gr.I 0.688 <0.08+ <0.01** 0.745 0.09+ <0.01** Gr.II - <0.04* <0.01** 0.093+ <0.05* <0.01** Gr.III - - <0.03* 0.736 0.812 <0.01** Gr.IV - - - <0.01** <0.05* 0.589 Gr.V - - - - <0.07+ <0.01** Gr.VI - - - - - <0.04* Gr.VII - - - - - - 95
    • Highly significant decrease are seen in Group IV and VII when compared to others. Graph: 6 Shows the body weight in groups Body weight Chart 15 10 5 3rd day % change 0 6th day 9th day -5 12th day -10 14th day Grp I Grp Grp Grp Grp V Grp Grp II III IV VI VII Groups Apparent body weight gain is seen in control group. By 3rd day all the Groups II,III,IV,and V have a considerable loss of weight. Group II and V have a negligible increase by 6th day. Group III and VI have a considerable decrease in % of weight. Suggestive significant decrease is seen groups IV ,VI and VII by 9th day. All the groups show a significant increase by 12th -14th day except group VII as they were all dead by these days. 2. MORTALITY OF THE RATS RECORDED :- In groups I,II,III,V, &VI no mortality was observed until the last day (14thday).All these animals were then sacrificed for collecting organ tissues on 14th day. Theremaining two groups of escalated doses i.e group IV and VII had the death of rats onparticular days with particular doses as shown below in the tables.Table 23: Showing the number and % of rats dead in the given doses inGroup IV (Shuddha Malla).Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14Dose 0.36 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 19.8 21.6 23.4 96
    • No.ofRats 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 2/8 3/8 7/8 7/8 7/8 7/8Dead% of 0 0 0 0 0 0 0 0 25 37.5 87.5 87.5 87.5 87.5deathTable 24: Showing the number and % of rats dead in the given doses ingroup VII (Ashuddha Malla)Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14Dose 0.36 1.8 3.6 5.4 7.2 9.0 10.8 12.6 14.4 16.2 18.0 19.8 21.6 23.4No.ofRats 0/8 0/8 0/8 0/8 0/8 0/8 1/8 3/8 8/8 ----- ----- ----- ----- -----Dead% of 0 0 0 0 0 0 12.5 37.5 100 ----- ----- ----- ----- -----deathGraph 7: Schematic representation of the Quantal dose response curve 97
    • Graph showing % death due to Shuddha Malla in ranging doses 100 90 87.5 87.5 87.5 7.5 8 80 % of death of rats 70 60 50 40 37.5 30 25 20 10 0 0 0 0 0 0 0 0 0 0.36 1.8 3.6 5.4 7.2 9 10.8 12.6 14.4 16.2 18 19.8 21.6 23.4 Dose mg/kg body weight Foot note:The red line shows the LD 50 and its dose margin.By the graphical method of Miller and Tainter190:- The dose when 3 rats were dead was:: 16.2 mg The dose when 7 rats were dead was:: 18.0 mg Therefore the dose for 4 rats i.e 50% of the rats to die is :- 18.0-16.2=1.8 mg When the next 4 rats die with an extra dose of 1.8 mg ::1.8÷4= 0.45 for a single rat.For 4 rats i.e 50% rats the corresponding probit is-16.2+0.45= 16.65mg is thepredictable LD50. If median efficient dose is taken as 5.4 mg as per dose-response curvethen: Therapeutic index:- LD50/ED50 16.65÷5.4= 3.08.Graph 8: Schematic representation of the Quantal dose responsecurve. 98
    • Graph showing % of death due to Ashuddha Malla in ranging doses 110 100 100 90 % of Death of rats 80 70 60 50 40 37.5 30 20 10 12.5 0 0 0 0 0 0 0 0.36 1.8 3.6 5.4 7.2 9 10.8 12.6 14.4 16.2 Dose mg/kg Body weight Foot note: The red line shows the LD 50 and its dose margin.By the graphical method of Miller and Tainter190:- The dose when 3 rats were dead was:: 12.6 mg The dose when 8 rats were dead was:: 14.4mg Therefore the dose for 4 rats i.e 50% of the rats to die is :- 14.4-12.6=1.8 mg When the next 5 rats die with an extra dose of 1.8 mg ::1.8÷5= 0.36 for a single rat.For 4 rats i.e 50% of rats the corresponding probit is -12.6+0.36= 12.96mg is thepredictable LD50. If median efficient dose is taken as 4.5 mg as per dose-response then: Therapeutic index:- LD50/ED50 12.96÷4.5= 2.88.STATISTICAL RESULTS OF BLOOD & SERUM PARAMETERS:-Table 25: Comparison of Hb between groups. 99
    • Group I Group II Group III Group IV Group V Group VI Group VII Hb P value Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1st 16.10 0.7 14.75 0.9 14.73 0.6 15.30 1.0 14.83 0.9 15.13 1.0 15.75 0.9 0.015*day 7th 15.14 1.1 13.49 1.4 14.04 1.6 15.69 1.3 14.33 1.1 14.20 1.2 14.91 1.6 0.037*day14th 13.35 1.7 11.74 0.9 15.61 1.3 16.50 15.05 0.8 15.95 1.5 - - <0.001**day%Change from 1st day At 7th +6.0 -8.6 -4.7 +2.5 -3.4 -6.1 -5.3 -day At14th +17.1 -20.4 +6.0 +7.8 -1.5 +5.5 - -day P F=17.501 F=26.712 F=6.957 F=1.776 F=3.982 t=1.326 - -value P=<0.001 P=<0.001 P=0.008 P=0.205 P=0.043 P=0.226 Group I shows a marginal increase by 7th day and a significant inrease in Hb% by 14th day. The Group II shows negligible decrease from 1st -7th day and then from 7th -14th day a suggestive significant decrease. In group III there is negligible decrease from 1st-7th day,there is a negligible increase in % change from 7th-14th day. In group IV there is negligible increase in Hb% by 14 days. In group V there is a negligible decrease from 1st-14th day. In group VI though there is negligible decrease in 7 days then a negligible increase by 14th day. In group VI I there is negligible decreasing of Hb% from 1st -7th day.Table 26:Comparison of TLC between groups. Group I Group II Group III Group IV Group V Group VI Group VIITotal Pcount value Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD1st 11600 961 10525 843 11600 711 11468 1037 10837 1225 11312 1135 11500 1177 0.262day 100
    • 7th 10412 1129 10118 1005 10718 980 10450 892 10631 1057 10018 815 10437 1067 0.795 day14th 10237 1315 9818 939 10575 1045 9500 10843 707 10237 582.9 - - 0.325day%Change from 1st dayAt 7th -10.2 -3.9 -7.6 +58.9 -1.9 -11.4 +79.2 - day At14th -11.7 +6.7 -8.8 +17.2 +0.1 -9.5 - -day P F=16.066 F=4.699 F=6.344 F=0.296 F=8.042 t=2.653 - -value P=<0.001 P=0.058 P=0.011 P=0.748 P=0.005 P=0.033 In Group I TLC is negligibly reduced by 14th day. In group II TLC % is negligibly decreased and increased by 14th day. In group III TLC % is negligibly decreased. In group IV TLC% is significant increase by 7 days and by 14th day it is increased significantly. In group V the % of TLC has a negligible decrease. In group VI the TLC % has decreased negligibly. In group VII there is highly significant increase in 7 days.Table 27: Comparison of Differential Counts-Polymorphs betweengroups. Group I Group II Group III Group IV Group V Group VI Group VII P DC-P value Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1st day 19.38 5.3 23.13 2.2 23.63 4.5 24.50 3.0 23.25 2.2 24.13 4.9 23.13 2.7 0.161 7th day 26.00 4.2 24.38 3.8 23.50 4.8 26.75 3.6 23.00 3.9 24.13 2.2 25.50 4.2 0.43514th day 27.38 4.4 22.63 2.7 24.38 3.1 26.00 25.00 3.7 28.75 5.6 - - 0.062+%Change from 1st day 101
    • At 7th +34.2 5.4 -0.5 9.2 -1.1 0.0 10.3 - day At 14th +41.3 -2.2 3.2 6.1 7.5 19.2 - - day F=9.793 F=0.919 F=0.131 F=1.047 F=3.434 t=1.239P value - - P=0.002 P=0.422 P=0.878 P=0.377 P=0.061 P=0.255 In group I there is moderately significant increase by 7th and by 14th day. In group II there is negligible increase in 7 days but later a negligible decrease. In group III there is no change but later there is a negligible increase. In group IV there is a negligible increase by 7th and 14th day.. In group V there is no change in 7 days but by 14th day there is a negligible increase. In group VI there is no change absolutely but there a significant increase by 14th day. In group VII there is a negligible increase in 7 days.Table 28:Comparison of Lymphocytes between groups. Group I Group II Group III Group IV Group V Group VI Group VIIDC- P L value Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1st 81.38 5.7 73.88 1.9 77.63 5.3 76.25 5.4 74.13 2.5 77.38 6.4 76.00 4.2 0.052+day 7th 69.63 5.0 71.00 4.1 73.38 4.1 70.50 4.3 72.13 3.4 72.00 1.3 71.38 4.6 0.616day14th 71.00 2.4 68.75 5.8 72.75 2.0 80.00 71.00 4.2 76.13 7.9 0.050+day%Change from 1st day At 7th -1.4 -3.9 -5.5 -7.5 -2.7 -6.9 -6.1 -day At14th -1.7 -6.9 -6.3 14.9 -4.2 -11.6 - -day P F=13.684 F=4.807 F=3.926 F=2.264 F=1.949 t=2.499 - -value P=0.001 P=0.026 P=0.044 P=0.141 P=0.179 P=0.041 In group I , II & III there is negligible decrease in lymphocyte % from 1st to 14th day. 102
    • In group IV negligible decrease by 7 days then later 7 days the % change has increased significantly. In group V the DC-L% has decreased slightly from 1st to 14th day. In group VI the % of DC-L has decreased negligibly. In group VII the % change of DC-L is negligibly decreased in 7 days.Table 29:Comparison of Eosinophils between groups. Group I Group II Group III Group IV Group V Group VI Group VIIDC- P E value Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1st 1.50 1.2 1.13 0.8 1.00 0.5 1.00 0.9 0.88 0.6 1.13 0.6 1.13 0.6 0.819day 7th 1.63 1.2 1.88 0.8 1.88 1.4 1.00 0.9 2.50 1.1 2.13 1.2 1.50 1.4 0.257day14th 1.13 1.1 1.50 0.5 1.00 0.8 2.00 1.38 0.5 1.38 0.5 - - 0.627day%Change from 1st day At 7th +8.3 +66.7 +7.5 0.0 +85.7 +88.9 +33.3 -day At14th -25.0 +33.3 0.0 -80.0 +57.1 +22.2 - -day P F=0.532 F=4.807 F=3.926 F=9.404 F=3.138 t=0.629 - -value P=0.001 P=0.026 P=0.044 P=0.003 P=0.078 P=0.549 In group I there is a negligible increase by 7th day, significant decrease by 14th day. In group II there is a moderately significant increase by 14th day. In group III there is a negligible increase by 7 days . In group IV there is highly significant increase by 14th day. In group V there is highly significant increase which moderately increases by 14th day. In group VI there is highly significant increase by 7th day,a suggestive significant increase by 14th day. In group VII there is moderately significant increase by 7 days.Table 30:Comparison of Monocytes between groups. 103
    • Group I Group II Group III Group IV Group V Group VI Group VII PDC-M value Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD1st day 2.25 1.0 2.38 1.3 2.25 0.9 1.63 0.7 2.00 1.5 2.50 0.9 1.75 0.9 0.6197th day 2.25 1.0 1.38 1.3 1.38 1.8 1.63 1.2 1.38 1.1 1.38 1.4 0.88 0.6 0.531 th 14 2.25 1.4 1.00 0.8 1.13 1.0 - - 1.50 0.9 2.38 1.9 - - 0.114 day%Change from 1st dayAt 7th 0.0 -42.1 -38.9 +5.0 -31.3 -45.0 -50.0 - dayAt 14th 0.0 -57.9 +50.0 - -25.0 -5.0 - - day P F=0.000 F=3.158 F=2.122 F=0.605 F=1.231 t=2.966 - -value P=1.000 P=0.074 P=0.157 P=0.560 P=0.332 P=0.021 In group I there is no change absolutely. In group II there is moderately significant decrease from 1st-14th day. In group III there is moderately significant decrease by 7th day ,a moderately significant increase by 14th day. In group IV there is negligible increase by 7 days. In group V there is significant decrease by 14th day. In group VI there is moderately significant decrease by 7th day. In group VII there is moderately significant decrease by 7th day.Table 31:Comparison of Packed Cell Volume between groups. Group I Group II Group III Group IV Group V Group VI Group VII PPCV value Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1st 49.71 3.9 45.78 2.3 47.10 5.0 49.43 6.4 48.03 5.4 48.26 5.9 50.84 4.4 0.465day 7th 44.41 5.9 41.86 4.3 43.44 4.0 43.63 4.7 42.98 3.3 42.65 3.6 45.03 4.2 0.823day14th 41.73 5.9 39.45 4.6 43.76 5.6 49.20 44.80 3.6 43.71 3.6 - - 0.175day%Change from 1st day 104
    • At7th +10.7 -8.5 -7.8 -11.7 -10.5 -15.6 -11.4 -day At14th +16.1 -13.8 +7.1 -0.5 -6.7 -9.4 - -day P F=11.557 F=3.544 F=4.948 F=2.520 F=6.261 t=3.077 - -value P=0.001 P=0.001 P=0.024 P=0.116 P=0.011 P=0.018 In group I has increased significantly by14th day. In group II % has decreased negligibly. In group III negligible decrease by 7th day and a negligible increase by 14th day. In group IV there is a significant decrease in 7 days. In group V there is a negligible decrease by 14th day. In group VI there is significant decrease by 7th day. In group VII a significant decrease is seen by 7 days.Table 32:Comparison of Red blood Corpuscles between groups. Group I Group II Group III Group IV Group V Group VI Group VIIRBC P value Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1st 5.93 0.3 5.44 0.5 5.39 0.5 6.01 0.6 5.41 0.6 5.61 0.9 5.97 0.9 0.204day 7th 4.92 0.5 4.77 0.7 4.86 0.6 5.04 0.4 4.77 0.4 4.73 0.4 5.14 0.5 0.581day14th 4.55 0.7 4.13 0.6 5.95 1.0 5.38 5.53 0.5 6.31 1.1 - - <0.001**day%Change from 1st day At 7th +16.9 +12.4 -9.9 -16.0 -11.9 -15.7 -13.8 -day At14th +23.3 -4.1 10.4 -10.4 2.1 -12.4 - -day P F=29.437 F=16.829 F=6.105 F=5.875 F=5.210 t=1.999 - -value P<0.001 P=0.001 P=0.002 P=0.014 P=0.050 P=0.086 In group I there is significant increase by14th day. In group II though there is significant increase by 7 days. 105
    • In group III there is negligible decrease by 7th day, and a negligible increase by 14th day. In group IV there is significant decrease by 7th day. In group V there is suggestive significant decrease by 7th day . In group VI there is significant decrease from 1st-14th day. In group VII there is significant decrease in 7 days. Table 33:Comparison of Urea between groups. Group I Group II Group III Group IV Group V Group VI Group VII P valueUrea Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1st 29.13 2.1 31.81 4.5 40.36 5.6 29.41 2.0 31.59 4.1 36.04 8.1 33.09 5.6 0.001**day7th 39.78 9.5 30.44 5.4 42.85 11.6 55.40 11.5 34.15 5.5 59.26 17.9 56.49 13.4 <0.001**day14th 39.73 10.6 34.01 3.7 45.23 11.8 69.90 33.40 5.9 57.05 19.5 - - 0.001**day%Change from 1st day At 7th 36.5 -4.3 6.2 88.4 8.1 64.4 70.7 -day At14th 36.4 6.9 12.0 97.7 5.7 58.3 - -day P F=4.063 F=1.425 F=1.497 F=0.520 F=8.710 t=5.285 - -value P=0.045 P=0.273 P=0.261 P=0.606 P=0.003 P=0.001 In group I there is moderately significant increase by 14th day. In group II it has decreased negligibly by 7 days. In group III negligible increase by 14th day. In group IV highly significant increase by 7th , 14th day. In group V the negligible increase by 14th day. In group VI increased moderately by14th day. In group VII there is highly significant increase by 7th day. 106
    • Table 34:Comparison of Creatinine between groups. P Group I Group II Group III Group IV Group V Group VI Group VII value Creatinine Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1st day 0.87 0.3 1.00 0.4 1.03 0.3 1.35 0.4 0.67 0.1 0.91 0.5 0.77 0.4 0.011* 7th day 0.73 0.2 0.87 0.3 0.66 0.2 0.72 0.1 0.66 0.1 0.66 0.2 0.72 0.2 0.371 14th day 0.69 0.2 0.59 0.1 0.77 0.2 0.82 0.68 0.1 0.76 0.3 - - 0.473 %Change from 1st day At 7th day -1.3 12.9 35.2 46.7 1.9 26.9 6.3 - At 14th -2.6 40.5 25.2 39.1 2.2 16.4 - day F=0.868 F=6.487 F=6.144 F=0.104 F=2.180 t=0.388 P value - - P=0.445 P=0.010 P=0.012 P=0.902 P=0.150 P=0.709 In group I there is a negligible decrease by 14 days. In group II there is moderate increase in 7 days but a significant increase in 14 days. In group III there is moderately significant increase seen by 14th day. In group IV there is moderately significant increase by 14 days. In group V there is negligible increase by 14th day. In group VI there is moderately significant increase by 14th day. In group VII there is negligible increase by 7 days. Table 35 :Comparison of SGOT between groups Group I Group II Group III Group IV Group V Group VI Group VIISGOT P value Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD1st day 51.50 8.6 63.50 9.2 59.13 10.2 61.88 8.5 48.50 8.8 56.25 10.3 52.75 8.6 0.016*7th day 51.63 5.3 67.75 18.8 49.63 6.8 83.50 6.8 48.50 3.7 67.75 16.8 86.63 11.3 <0.001** 14th 53.88 7.5 53.50 11.9 54.63 6.5 86.00 54.13 5.3 73.00 20.0 - - 0.002** day%Change from 1st dayAt 7th 0.2 +6.7 +6.1 +14.9 +9.0 +20.4 +64.2 - dayAt 14th 4.6 +5.7 +7.6 +19.0 +11.6 +29.8 - - day 107
    • P F=0.278 F=2.747 F=6.250 F=3.115 F=8.041 t=7.725 - -value P=0.761 P=0.099 P=0.034 P=0.076 P=0.005 P<0.0001 In group I negligible increase is seen. In group II there is negligible increase by 14th day. In group III there is negligible increase by 14th day In group IV there is suggestive significant increase in % by 14th day. In group V there is suggestive significant increase by 14th day. In group VI there is suggestive significant increase seen by 14th day. In group VII there is moderately significant % change increased by 7th day. Table 36:Comparison of SGPT between groups. Group I Group II Group III Group IV Group V Group VI Group VII SGPT P valu Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 1st day 155.25 13.1 167.88 8.4 163.50 8.5 173.88 11.6 177.13 21.2 170.75 4.2 170.13 8.0 0.015* 7th day 163.63 25.0 201.13 9.8 164.75 6.9 186.75 3.6 209.00 19.8 192.25 25.9 187.25 17.8 <0.001 14th 165.88 24.5 171.50 17.7 171.75 8.7 24.13 68.2 217.50 34.0 195.13 33.1 - - <0.001 day %Change from 1st day At 7th +0.4 +9.8 +0.8 +7.4 +18.0 +12.6 +30.1 - day At 14th +1.8 +2.2 +5.0 +86.1 +22.8 +14.3 - - day F=1.677 F=19.888 F=2.066 F=6.889 F=4.951 t=2.124 P value - - P=0.222 P<0.001 P=0.164 P=0.025 P=0.024 P=0.071 In group I there is a negligible increase. In group II there is negligible increase. In group III there is negligible increase. In group IV there is negligible increase by 7th day,a highly significant increase by 14th day. In group V moderately significant change seen in 14 days. In group VI moderate increase is observed for 14 days. In group VII highly significant increase in is obtained. 108
    • Table 37:Shows the HISTOPATHOLOGY RESULTS:-G HEART BRAIN LIVER KIDNEY LUNGS SPLEEN GASTRR -ICO MUCOU —SAPI Normal Normal Normal Normal Mild Normal Normal Congestion OedemaII Normal Normal Normal Normal ---same----- Normal NormalII Normal Normal Normal Normal -----same----- Normal Mild LesionII Destruction Mild Mild Mild Oedema, Mild MildV Of cardiac Congestion, fatty congestion Haemorrhage congestion keratiniz- Fibres. Degenerating infiltrations Degeneration, Infiltration of ation,desc Neurons. severe inflammatory -umation haemorrhage. cells in of krypts interstitialV Normal Normal Normal Normal Mild congn Normal NormalV Congestion Mild lesion , Congestion Atrophic Severe Normal Villi Of cells. haemorrhage Of cells Glomeruli, haemorrhage showingI Periportal Tubular around the descumat infiltration Changes, vessel,thicken -ion necrosis -ing of ,destructi alveolar wall. -onV Severe Congestion Destruction Congestion, Periartiritis, Congestion DescumatII haemorrhag Mild swollen Of Haemorrhage Thickening of haemorrha -ion due -e & oligodendrites hepatocytes Necrosis of alveolar ge,prolifera to destruction Infiltration of Remnants Tubular wall,haemorr tion of irritation. of muscle Lymphocytes. Of epithelium hage lymphoblas fibres. Epithelium. with ts around proteinacious the casts,swollen arteriole. tubules. 109
    • SUB-ACUTE STUDYSTUDY DESIGN: Single dose and repeated dose toxicity study within 5 groups eachwith 8 animals is undertaken to study the toxicity based on different parameters.. Group 1: Control Group 2: Single therapeutic dose of Shuddha Malla only on 1st day and 28 days observation. Group 3: Single therapeutic dose of Shuddha Malla on all 28 days. Group 4: Single therapeutic dose of Ashuddha Malla only on 1st day and 28 days observation. Group 5: Single therapeutic dose of Ashuddha Malla on all 28 days. GENERAL OBSERVATIONS: The group 3 and 5 showed an improved alertness compared to other groups. Increased motor activity is observed in groups 3 and 5. The Pinna reflex was normal in all groups. The colour change in faecal matter from straw yellow to coffee brown was observed by 16th and 18th days in group 5 and 3 respectively. RESULTS OF THE BODY WEIGHT:Table 38: Comparison of weight between groups. Group I Group II Group III Group IV Group V Body P value weight Mean SD Mean SD Mean SD Mean SD Mean SD0th day 153.63 4.57 156.00 4.54 152.63 2.33 156.63 12.41 153.50 2.62 0.6793rd day 158.50 6.55 157.00 6.87 147.25 3.54 155.75 14.08 157.38 5.21 0.059+6th day 171.88 8.37 167.13 9.36 153.88 2.36 163.75 16.54 165.88 4.49 0.011*9th day 172.00 7.72 165.67 9.65 156.00 3.46 166.50 20.58 161.50 3.89 0.16612th day 181.50 7.99 179.17 14.48 171.17 3.19 185.67 22.50 176.00 5.93 0.38815th day 184.50 12.45 187.50 17.82 175.75 7.89 182.75 2.87 183.50 3.70 0.2018th day 202.00 18.17 202.25 18.45 187.75 13.15 198.75 2.75 200.00 5.16 0.53321st day 213.25 25.25 204.50 18.08 202.75 13.57 201.00 13.22 206.50 4.80 0.85124th day 240.50 45.96 207.00 26.87 212.00 0.00 214.50 7.78 208.50 2.12 0.651 110
    • 27th day 216.50 40.31 214.00 28.28 214.00 11.31 218.50 26.16 217.00 7.07 1.000% Change from 0th day3rd day 3.17 -0.64 -3.52 -0.56 -2.52 -6th day 11.88 7.13 -0.82 4.55 -8.06 -9th day 11.96 6.20 2.21 6.30 -5.21 -12th day 18.14 14.85 12.15 18.54 14.66 -15th day 20.10 20.19 15.15 16.68 19.54 -18th day 31.49 29.65 23.01 26.90 30.29 -21st day 38.81 31.09 32.84 28.33 34.53 -24th day 56.55 32.69 38.90 36.95 35.83 -27th day 40.93 37.18 40.21 39.51 41.37 - Graph 9: Schematic representation of differences in body weight. Body weight Chart 60 50 3rd day 40 % Change 30 6th day 20 9th day 10 12th day 0 15th day -10 -20 18th day 21st day Grp I Grp II Grp III Grp IV Grp V 24th day Groups 27th day Normal body weight gain is seen in Group I. On 3rd day there is a considerable suggestively significant decreased percentage of weight loss in Groups II, III,IV and V. Group II when compared to III, IV and V has a significant increase in body weight by 6th day. Group III the significant increase is seen only by 9th day. Group IV also shows significant increase from 6th day but to lesser percentage compared to Group II. Group V has suggestive significant decrease until 9th day then by 12th day a significant increase is shown. 111
    • STATISTICAL RESULTS OF BLOOD & SERUM PARAMETERSTable 39: Comparison of Hb between groups. Group I Group II Group III Group IV Group V Hb P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 15.1 0.6 16.5 1.2 16.7 0.4 16.7 0.4 16.4 0.8 0.001**7th day 13.9 0.4 15.7 1.2 15.7 0.4 15.6 0.4 15.5 0.7 <0.001**14th day 12.9 1.2 13.4 0.9 20.8 1.3 14.9 1.0 13.0 1.2 0.009**21st day 14.2 0.5 14.3 0.6 24.6 0.7 14.7 0.2 13.6 0.2 0.041*28th day 14.8 0.4 14.0 0.1 13.9 1.4 14.4 0.3 23.7 1.1 0.480% Change from 1st dayAt 7th day -8.2 -4.5 -6.2 -6.6 -5.0 -At 14th day -14.9 -18.6 +7.2 -10.4 -20.9 -At 21st day -6.5 -13.1 +12.9 -11.6 -16.9 -At 28th day -2.6 -15.2 +7.1 -13.5 +16.3 - In group I there is suggestive decrease in % of Hb. In group II also a suggestive significant decrease is seen from 7th -28th day. In group III a moderate increase is seen by 21st day. In group IV there is a significant decrease from 7th-28th day. In group V there is significant Hb% decrease until 21st day but later by 28th day there is a significant increase.Table 40: Comparison of Total count between groups. Group I Group II Group III Group IV Group VTotal P valuecount Mean SD Mean SD Mean SD Mean SD Mean SD1st 8987.5 1035.7 10737.5 1536.2 10425.0 554.8 11325.0 660.6 10587.5 653.4 <0.001**day7th 8125.0 1203.3 10012.5 1652.2 10168.8 459.0 10525.0 406.2 10156.3 550.0 <0.001**day14th 8750.0 1385.3 9041.7 1774.9 10600.0 720.4 10383.3 1162.2 8200.0 1714.4 0.025*day21st 12900.0 2891.4 10387.5 611.5 9850.0 - 10600.0 679.5 9150.0 669.6 0.056+day28th 8500.0 141.4 9075.0 813.2 7550.0 919.2 10550.0 989.9 10550.0 989.9 0.055+day 112
    • % Change from 1st dayAt7th -9.6 -6.8 -2.5 -7.1 -4.1 -dayAt14th -2.6 -15.8 +1.7 -8.3 -22.6 -dayAt21st +43.5 -3.3 -5.5 -6.4 -13.6 -dayAt28th -5.4 -15.5 -27.6 -6.8 -0.4 -day In group I there is moderate decrease by 14th day but a moderate significant % increase is seen by 21st day and again a slight decrease by 28th day. In group II there is a moderately significant decrease from 7th to 28th day. In group III though by 7th day there is marginal decrease and a moderate decrease by 28th day. In group IV a negligible decrease is seen. In group V significant decrease is observed by 14th and 21st day.Table 41: Comparison of DC-P between groups. Group I Group II Group III Group IV Group V DC-P P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 51.1 4.4 57.0 6.0 58.5 4.3 58.4 3.2 53.9 5.6 0.016*7th day 48.3 5.4 53.4 6.1 49.5 5.7 50.0 8.1 53.4 1.7 0.27914th day 47.3 3.6 52.2 5.5 51.5 4.9 54.3 4.4 49.8 6.4 0.20121st day 43.0 4.4 49.0 7.4 52.8 6.4 45.8 9.6 39.0 2.9 0.078+28th day 47.5 7.8 53.0 1.4 46.5 2.1 50.0 14.1 55.5 0.7 0.722% Change from 1st dayAt 7th day -5.6 -6.4 -15.4 -14.3 -0.9 -At 14th day -7.4 -8.5 -12.0 -6.9 -7.5 -At 21st day -15.9 -14.0 -9.8 -21.6 -27.6 -At 28th day -7.1 -7.0 -20.5 -14.3 +3.0 - In group I & II there is negligible change in polymorphs % but significant by 21st day. 113
    • In group III significance decrease is seen by 28th day. In group IV decrease is significant by 21st day. In group V the decrease is significant by 21st day but by 28th day there is a negligible increase.Table 42: Comparison of DC-L between groups. Group I Group II Group III Group IV Group V DC-L P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 56.4 3.2 46.9 7.2 77.6 5.3 52.8 2.6 48.4 2.6 <0.001**7th day 52.0 3.0 49.3 6.1 48.8 6.7 48.3 6.4 43.3 2.5 0.035*14th day 48.8 4.2 46.0 7.6 47.7 5.3 45.3 5.0 45.0 7.8 0.79021st day 55.5 3.5 49.5 5.8 45.3 7.1 52.0 10.0 60.8 3.0 0.037*28th day 53.0 9.9 46.0 2.8 46.5 6.4 46.0 14.1 43.0 1.4 0.809% Change from 1st dayAt 7th day -7.8 +5.1 -37.2 -8.5 -10.6 -At 14th day -13.4 -1.9 -38.6 -14.1 -7.0 -At 21st day -1.6 +5.6 -41.7 -1.4 +25.6 -At 28th day -6.0 -1.9 -40.1 -12.8 -11.1 - In group I there is a negligible decrease in lymphocyte %. In group II there is negligible increase and later by 21st day there is a negligible increase. In group III there is a moderately significant decrease by 7th ,14th,21st and 28th days. In group IV there is a negligible decrease by 7th day,a significant decrease by 14th and 28th day. In group V negligible decrease is seen by 14th day but later by 21st day a significant increase is seen.Table 43: Comparison of DC-E between groups.DC-E Group I Group II Group III Group IV Group V P value 114
    • Mean SD Mean SD Mean SD Mean SD Mean SD1st day 2.6 1.5 1.1 0.8 1.0 0.5 2.0 1.3 1.4 0.9 0.025*7th day 0.9 1.0 1.1 0.6 1.6 0.7 1.1 0.6 1.4 0.9 0.41214th day 0.5 0.5 1.2 1.2 1.2 1.2 0.7 0.8 1.7 0.5 0.20121st day 0.5 0.6 1.3 1.5 0.8 1.0 0.8 1.0 1.3 1.0 0.79028th day 0.5 0.7 0.5 0.7 1.5 2.1 1.0 1.4 0.5 0.7 0.898% Change from 1st dayAt 7th day -66.7 -0.0 +62.5 -43.8 -70.0 -At 14th day -81.0 +3.7 +16.7 -66.7 +21.2 -At 21st day -81.0 +11.1 -25.0 -62.5 -9.1 -At 28th day -81.0 -55.6 +50.0 -50.0 -63.6 - In group I a moderately significant decrease is observed on 7th,14th,21st,28th days. In group II there is no or negligible change seen by 21st day but a moderately significant decrease is seen by 28th day. In group III there is a moderately significant increase by 7th day but later by 21st day a significant decrease and a moderate increase by 28th day. In group IV there is a moderate decrease on all 7th ,14th,21st and 28 days. In group V there is a highly significant decrease by 7th day but by 14th ,21st day a negligible increase and a moderately significant decrease by 28th day.Table 44: Comparison of DC-M between groups. Group I Group II Group III Group IV Group V DC-M P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 2.6 1.1 2.4 1.3 1.4 0.7 1.3 1.0 1.3 1.0 0.023*7th day 1.6 0.5 1.8 1.0 2.9 1.6 1.8 0.5 2.4 0.7 0.070+14th day 2.2 0.8 1.5 0.5 1.2 1.0 1.0 0.9 1.8 0.4 0.073+21st day 1.3 1.0 0.8 0.5 2.0 0.8 2.0 0.8 1.0 0.8 0.13328th day 1.5 0.7 1.0 1.4 1.5 0.7 2.0 0.0 1.5 0.7 0.830% Change from 1st day 115
    • At 7th day -38.1 -26.3 +100.1 +40.0 +90.0 -At 14th day -17.5 -36.8 -15.2 -20.0 +46.7 -At 21st day -52.4 -68.4 +45.5 +60.0 -20.0 -At 28th day -42.9 -57.9 +9.1 +60.0 +20.0 - In group I is moderately % decreased by 7th ,21st,28th days. In group II significant decrease by 7th day and moderately significant decrease by 14th,21st and 28th day. In group III a highly significant increase is seen in % by 7th day and a negligible decrease by 14th day,a moderate increase by 21st day,negligible increase by 28th day. In group IV a moderately significant increase is seen by 7th day,21st and 28th day but a suggestive decrease is found by 14th day. In group V there is highly significant % increase by 7th day,moderate increase by 14th,a significant decrease by 21st,a significant increase by 28th day.Table 45: Comparison of RBC between groups. Group I Group II Group III Group IV Group V RBC P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 7.3 1.0 8.7 0.9 9.2 0.3 9.3 0.5 8.7 0.5 <0.001**7th day 6.0 0.5 6.0 0.5 8.4 0.4 7.9 0.9 7.5 0.7 <0.001**14th day 5.9 0.8 6.6 1.1 6.6 0.7 6.6 0.7 5.9 0.9 0.35021st day 6.0 0.8 6.5 0.6 6.8 0.5 6.4 0.6 5.1 0.1 0.005**28th day 5.3 1.0 6.5 0.7 6.3 0.4 7.0 0.2 40.5 2.1 <0.001**% Change from 1st dayAt 7th day -17.6 -30.4 -9.1 -14.9 -14.2 -At 14th day -19.6 -23.5 -28.3 -28.6 -31.8 -At 21st day -18.9 -25.3 -26.0 -30.8 -41.4 -At 28th day -27.8 -25.4 -31.4 -24.8 -85.9 - In group I there is a suggestive significant decrease % of RBC. In group II there is a significant decrease in RBC %. 116
    • In group III a negligible decrease by 7th day but a significant decrease by 14th,21st,28th day. In group IV a significant decrease is seen by 7th,14th,21st and 28th day. In group V a moderate decrease is seen by 21st day ,a highly significant decrease by 28th day.Table 46: Comparison of PCV between groups. Group I Group II Group III Group IV Group V PCV P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 43.7 2.6 48.8 1.6 51.4 2.9 50.7 2.2 49.3 1.5 <0.001**7th day 41.5 0.6 46.7 1.5 46.6 1.2 46.7 1.4 46.3 2.0 <0.001*14th day 39.6 5.1 44.0 3.1 40.6 1.1 57.4 9.5 38.3 3.0 <0.001**21st day 45.1 4.8 46.3 3.6 46.6 5.6 48.4 4.5 40.3 1.7 0.13628th day 45.0 7.1 42.5 2.1 41.0 1.4 45.5 0.7 40.5 2.1 0.556% Change from 1st dayAt 7th day -5.1 -4.3 -9.4 -7.9 -6.1 -At 14th day -9.4 -9.9 -21.1 +13.2 -22.2 -At 21st day +3.2 -5.3 -9.5 -4.5 -18.3 -At 28th day +2.9 -13.0 -20.3 -10.3 -17.8 - In group I a negligible increase and decrease % of PCV. In group II a negligible decrease is seen in PCV %. In group III a negligible decrease is seen by 7th and 21st day,a significant decrease by 14th and 28th day. In group IV a negligible decrease is seen by 7th ,21st,28th and a significant increase by 14th day. In group V a negligible decrease is seen by 7th day,a significant increase by 14th,21st and 28th day.Serum analysis:-Table 47: Comparison of SGOT between groups. SGOT Group I Group II Group III Group IV Group V P value 117
    • Mean SD Mean SD Mean SD Mean SD Mean SD1st day 160.0 27.0 165.1 18.5 166.5 20.1 166.7 15.4 167.0 25.0 0.9657th day 226.9 92.1 211.0 11.5 226.9 92.1 196.5 61.4 200.5 58.0 0.85014th day 105.8 77.7 159.6 28.3 161.9 19.3 168.1 9.6 105.8 77.7 0.094+21st day 135.3 33.9 134.9 32.1 182.1 12.0 182.1 12.0 144.9 22.8 0.061+28th day 136.7 22.7 119.7 12.6 161.2 74.9 113.7 3.7 94.4 6.3 0.485% Change from 1st dayAt 7th day 41.8 27.8 36.3 17.8 20.0 -At 14th day -33.9 -3.4 -2.8 0.8 +36.6 -At 21st day -15.4 -18.3 +9.4 9.3 +13.2 -At 28th day -14.6 +7.5 +.2 -31.8 +43.5 - In group I a moderately significant increase is seen by 7th day, then a moderate decrease by 14th day,a significant decrease by 21st and 28th day. In group II a suggestive significant increase is seen by 7th day but a negligible decrease by 14th day,a suggestive significant decrease by 21st and 28th day. In group III a moderately significant increase is seen by 7th day,a negligible decrease by 14th and 28th day,a negligible increase by 21st day. In group IV a suggestive significant increase by 7th day,a negligible decrease by 21st day,a moderately significant decrease by 28th day. In group V a suggestive increase in change by 7th day and 21st day,a moderate significant increase by 14th day and 28th day.Table 48: Comparison of SGPT between groups. Group I Group II Group III Group IV Group V SGPT P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 45.1 5.6 43.0 6.3 49.8 7.4 43.1 9.6 50.8 13.5 0.2947th day 69.6 2.8 52.0 15.1 69.6 2.8 79.8 21.2 91.7 3.5 <0.001**14th day 53.5 1.5 56.2 3.7 65.5 3.6 72.3 7.0 73.4 5.0 <0.001**21st day 42.3 6.6 42.3 6.6 77.5 5.2 73.7 18.0 72.4 4.3 <0.001**28th day 9.6 12.5 9.6 12.5 5.3 2.5 3.9 12.5 84.6 3.4 0.912 118
    • % Change from 1st dayAt 7th day 54.2 20.9 39.9 85.0 80.7 -At 14th day 18.6 30.7 31.7 67.7 44.6 -At 21st day -6.3 -1.7 55.8 70.9 42.7 -At 28th day -78.8 -77.7 69.3 90.9 91.0 - In group I a moderate increase in SGPT by 7th day, a suggestive significance by 14th day, a negligible decrease by 21st day,a highly significant decrease in % by 28th day. In group II a suggestive significant increase by 7th and 14th day, a highly significant decrease is seen by 28th day. In group III a moderately significant increase by 7th, 14th ,21st,28th day. In group IV a highly significant increase % is seen by 7th day,a moderately significant increase by 14th and 21st day,a highly significant increase by 28th day. In group V a highly significant increase by 7th day,a moderate increase by 14th and 21st, a highly significant increase by 28th day.Table 49: Comparison of Albumin between groups. Group I Group II Group III Group IV Group V Albumin P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 4.3 0.8 43.0 6.3 4.8 0.7 48.4 0.8 4.9 0.9 <0.001**7th day 3.3 1.1 3.3 1.1 6.6 1.0 5.6 1.0 6.4 1.3 <0.001**14th day 6.3 0.9 5.1 0.9 5.2 0.5 6.0 0.9 6.1 1.3 0.12821st day 5.6 0.7 3.8 1.9 4.5 0.3 5.1 1.0 3.1 2.3 0.17028th day 4.8 0.2 4.8 0.2 5.6 0.7 5.5 0.2 3.0 1.3 0.070+% Change from 1st dayAt 7th day -22.2 -92.3 38.1 -96.4 30.4 -At 14th day 46.5 -88.1 7.5 -94.5 25.5 -At 21st day 30.6 -91.1 -5.6 -84.3 -36.6 -At 28th day 11.3 -88.9 15.5 -94.2 -38.3 - 119
    • In group I a suggestive decrease by 7th day,a moderate increase by 14th day,21st day, a significant increase by 28th day. In group II a highly significant decrease in albumin by 7th,14th,21st,28th days. In group III a moderate increase by 7th day,a negligible increase by 14th,a moderate increase by 28th day. In group IV a highly significant decrease is seen by 7th -28th day. In group V a suggestive increase by 7th and 14th day,a moderate decrease by 21st and 28th day.Table 50: Comparison of TPR between groups. Group I Group II Group III Group IV Group V TPR P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 7.9 1.8 8.7 2.6 7.2 1.5 9.0 1.8 9.1 2.9 0.3777th day 9.4 1.4 12.3 0.8 12.8 1.6 11.5 0.8 8.7 1.0 <0.001**14th day 10.8 2.6 11.6 1.6 12.6 1.7 13.4 1.6 13.8 1.8 0.065+21st day 5.9 1.9 6.7 2.3 8.8 5.2 7.1 2.4 - - 0.64028th day 11.6 0.8 11.5 1.9 10.3 8.5 12.6 0.9 14.6 0.6 0.732% Change from 1st dayAt 7th day 19.6 42.4 77.6 27.7 -3.6 -At 14th day 37.3 33.8 75.3 49.4 52.2 -At 21st day -25.7 -22.5 22.0 -20.7 -100.0 -At 28th day 46.8 32.4 43.3 40.2 61.4 - In group I a suggestive increase is seen by 7th day,a moderate increase by 14th day and 28th day but there seems to be a decreased % change by 21st day. In group II a moderate increase in % by 7th ,14th and 28th day and a significant decrease by 21st day. In group III a moderate increase by 7th day,a highly significant increase % by 14th day,a suggestive increase by 21st day,a moderate increase by 28th day. 120
    • In group IV a suggestive increase by 7th day,moderate increase by 21st day,a significant decrease by 21st day,a moderate increase by 28th day. In group V a negligible decrease in TPR% by 7th day, a moderate increase by 14th day,highly significant decrease by 21st day, moderate increase by 28th day.Table 51: Comparison of CRT between groups. Group I Group II Group III Group IV Group V CRT P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 0.7 0.0 1.3 0.8 1.0 0.5 1.2 0.6 0.9 0.5 0.2317th day 0.7 0.1 0.9 0.7 0.4 0.2 0.7 0.1 0.6 0.2 0.068+14th day 1.1 0.6 1.6 0.1 1.4 0.2 1.8 0.7 1.0 0.4 0.038*21st day 1.3 0.6 1.1 0.7 1.6 0.4 7.1 2.4 1.3 0.8 <0.001**28th day 3.7 0.0 23.6 0.6 48.9 6.9 33.9 0.1 34.7 0.7 0.462% Change from 1st dayAt 7th day -2.1 -31.3 -62.4 -44.3 -36.5 -At 14th day 8.5 28.5 38.9 41.2 8.9 -At 21st day 4.5 -15.5 59.8 74.1 36.4 -At 28th day 4.1 84.3 71.6 73.6 97.3 - In group I a negligible change is seen in CRT change. In group II a moderate decrease by 7th day, a suggestive increase by 14th day,a suggestive decrease by 21st day,a highly significant increase by 28th day. In group III a moderate decrease by 7th day,a moderate increase by 14th and 21st day,a nearly highly significant increase by 28th day. In group IV a moderate decrease by 7th day,a moderate increase by 14th day,a highly significant increase by 21st and 28th days. In group V a moderately significant decrease by 7th day,a moderate increase by 21st,highly significant increase by 28th day. 121
    • Table 52:Comparison of BUN between groups. Group I Group II Group III Group IV Group V BUN P value Mean SD Mean SD Mean SD Mean SD Mean SD1st day 53.4 13.9 67.8 17.5 61.0 7.5 55.9 14.4 62.5 15.5 0.2987th day 53.9 12.1 30.9 9.6 55.5 13.4 78.0 35.8 67.3 11.2 <0.001**14th day 93.2 43.9 101.5 104.2 155.6 18.3 101.5 104.2 63.0 41.2 0.29521st day 33.3 25.9 201.9 20.0 26.2 20.5 41.2 18.2 32.7 13.5 <0.001**28th day 127.0 1.4 40.6 21.9 37.5 17.7 113.0 17.0 40.0 16.9 0.006**% Change from 1st dayAt 7th day 0.9 -54.4 -8.9 39.5 77.6 -At 14th day 74.6 49.7 55.3 81.5 10.9 -At 21st day -37.7 97.9 -57.0 -26.3 47.6 -At 28th day 38.0 -40.2 -38.5 100.1 86.0 - In group I a negligible change by 7th day,a significant increase by 14th day, a moderate decrease by 21st day,a moderate increase by 28th day. In group II a moderate decrease by 7th day,a moderate increase by 14th day,a highly significant increase by 21st day,a moderate decrease by 28th day. In group III a negligible decrease by 7th day,a moderate increase by 14th day,a moderate decrease by 21st dayand 28th day. In group IV a moderate increase by 7th day,a highly significant increase by 14th day,a moderate decrease by 21st day, a highly significant increase by 28th day. In group V the % increase is highly significant by 7th day,a moderate increase by 21st day,a highly significant increase by 28th day. 122
    • Table 53: Shows the HISTOPATHOLOGY RESULTS :-HEART:GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAYI Normal Normal Normal NormalII Normal Normal Normal NormalIII Normal Normal Mild congestion Mild infiltrationsIV Normal Normal Normal NormalV Normal Mild congestion Normal Oedematous fibres,severe oedemaBRAIN:GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAYI Normal Normal Normal NormalII Normal Normal Normal NormalIII Normal Normal Normal Mild congestionIV Normal Normal Normal NormalV Normal Congestion of Mild Mild cells degenerative congestion,infiltration changes of few mononuclear cells. LIVER:GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAYI Normal Normal Normal NormalII Normal Normal Normal NormalIII Normal Normal Normal Fatty changesIV Normal Normal Normal Increased kupffer Cells.V Normal Congestion of Mild Congestion,haemorrhage cells degenerative Swollen & necrosed changes Hepatocytes,degenerationKIDNEY:GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAYI Normal Normal Normal NormalII Normal Normal Normal NormalIII Normal Normal Normal Mild congestionIV Normal Normal Normal NormalV Normal Normal Severe Necrosis,tubular congestion. changes.LUNG:GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAYI Mild Mild Mild Mild congestion congestion congestion congestionII Mild Mild Mild Mild 123
    • congestion congestion congestion congestionIII Mild Mild Mild Mild congestion congestion congestion congestionIV Mild Mild Mild Mild congestion congestion congestion congestionV Mild Mild Severe Oedema,mild congestion congestion congestion. emphysemaSPLEEN:GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAYI Normal Normal Normal NormalII Normal Normal Normal NormalIII Normal Normal Normal NormalIV Normal Normal Normal NormalV Normal Normal Normal Mild congestion, Haemorrhage.GASTRIC MUCOSA:GROUP 7TH DAY 14TH DAY 21ST DAY 28TH DAYI Normal Normal Normal NormalII Normal Normal Normal NormalIII Normal Normal Regenerative More Changes basophilic cells.IV Normal Normal Normal NormalV Normal Normal Keratinization Keratinization Slightly proliferating cells. 124
    • DISCUSSION“The great enemy of knowledge is not error, but inertness. All that we want is discussion;and then we are sure to do well, no matter what our blunders may be. One error conflictswith another, each destroys its opponent, and truth is evolved.” - Henry Thomas Buckle Any theories or observations emerged from exhaustive classical and experimentalstudy can only be accepted if there is a proper reasoning (tarka) of the results &observations. When we begin with doubts, we shall end with certainties. Therefore, thetheories and facts are put forth for discussion in front of the scientific community. Suchdiscussions and scholastic deliberations let the scientific fraternity to ameliorate byknowing both the theoretical and practical profiles about the work.So, here is a sagaciousdiscussion regarding the in and outs of present study entitled “Pharmaceutico-analyticaland toxicological study of Shuddha Malla on Wistar albino rats (An experimentalStudy)”. It is analyzed under 4 headings. • Review of literature. • Pharmaceutical study. • Analytical study. • Experimental study.Review of literature: Review of literature is discussed under following headings. ∗ Drug Review (Malla, Karavellaka). ∗ Review of Pharmaceutical Procedure. ∗ Analytical Review. ∗ Experimental Review.Drug Review: Malla is in use as a rasoushadhi since ages.Eventhough it is highlighted in majorRasashastra texts, it was first mentioned by Sushrutha as a therapeutic drug. Its wideapplications as medicine in shwasa, kushta, yakshma, sandhivata, phiranga etc., Itsactions as balya, vrushya, rasayana has given it the name. After all these merits Malla hasa synonym of Dhathu Visha/ Shankha Visha with its aushadhi mathra as 1/120th-1/30thof a ratti wherein the maaraka mathra is also mentioned cautiously to be 1 ratti.Thereforeenough care is taken during Malla matra nirmana as a safety concern.This gives a base tothink about the relationship of dose and toxicity. 125
    • Inorganic arsenic is a human poison. Organic arsenic is less harmful. Arsenic isan ultra-trace essential element (nonmetal). The deficiency signs are impairment ofgrowth, reproduction, heart function. The specific function is increased arginine leads tourea and ornithine which helps in metabolism of methyl compounds.After a very smalldose of arsenic most of the absorbed inorganic arsenic undergoes methylation, mainly inthe liver, to monomethylarsonic acid and dimethylarsinic acid which are excreted, alongwith residual inorganic arsenic in the urine. However, if the dose of arsenic is very large,the elimination half-life is prolonged. Once absorbed, arsenic rapidly combines with theglobin portion of haemoglobin and therefore localises in the blood. In one report fromChina the monotherapy of Arsenic trioxide produced a complete clinical response in 9 of10 with relapsed APL.Consequently, TRISENOX (As2 O3) was approved for the relapsedor refractory APL by US FDA in September 2000.This gives a clear picture of thecommendable updates from the modern research. Karavellaka (Momordica Charantia) belongs to Shaaka Varga,Cucurbitaceaefamily.It is a tiktha skandha dravya, widely grown for edible fruit, which is among themost bitter of all vegetables.Classically meant to be tridoshashaamaka.Its main actionsare said to be to kill bacteria,viruses,cancer cells,leukemia cells,prevents tumors,fightsfree radicals,cleanses blood,detoxification etc.,which would not only help the purificationprocedure but also enhance the drug qualities to which it comes into contact.Glycosides,Alkaloids, charantin, charine, hydroxytryptamines, lanosterol, rosmarinicacid, rubixanthin, spinasterol, steroidal glycosides,stigmasta-diols etc.Galactouronic acidwhich is found as a rare and special entity can complex with the toxic molecules for theorganization, may help in the obviating and eliminating the toxic properties of Mallawhich might be making the drug molecule assimilative and enhance for its prerogativeaction.Review of Pharmaceutical Procedure:- Pharmaceutical procedure adopted in the present study was Shodhana samskaraby “Swedana” • “Samskara” which defines itself as ‘Gunantharaadhaanam’ i.e enhancement of the qualities of a drug and ‘Doshaapanayanam’ i.e to remove the unwanted impurities. 126
    • • “Shodhana” is one of the initial norms to be instigated in any of Rasa preparations without which it remains unaccomplished. The word meaning may merely be the Purification but unto its depth it incarcerates the toxic substances and enhances the efficacy and safety of the drug molecule. The mode of each pharmaceutical procedures are unique in its own way. • “Swedana” is one of such ascendant procedures adopted which is used for shodhana of many Rasadravyas. In this process the drug is boiled in the liquids, which are ksharas, amlas or both, and medicinal juices, with the help of Dolayantra. Fluxation process would have occurred in this kind of Shodhana. Now a days some of the Rasadravyas are artificially prepared. So shodhana isindicated to eliminate all such toxic qualities, induce and enhance certain special qualitieswhich are essential for the easy assimilation of the material in the living body. • Dola Yantra and Khalva Yantra are used instruments which are the basic engineered techniques for purification method.Analytical Review: The Physico – Chemical tests like Ash value, Acid insoluble ash, Water insolubleash, Loss on ignition, Loss on drying and pH value are some of the physicochemicaltests. AAS for the estimation of Arsenic. AAS is a rapid method if only a few elementsare being tested. X-ray diffraction is a novel and a powerful technique in the elucidationof 3-dimensional structure, composition of any matter. Structural features can bedetermined at molecular and atomic level. It is an important technique for establishing thebatch-to-batch reproducibility of a crystalline form is X-ray Powder diffraction.ParticleSize analysis through laser diffraction technique is one of most upgraded technique whichhas a wide dynamic measuring range, Flexibility, Generation of volume-based particlesize distributions, Rapid data acquisition, Ease of Verification.Toxicological review: Toxicology traditionally known as the science of poisons began with earlycave dwellers who recognized poisonous plants and animals and used their extracts forhunting or warfare. Simultaneously, with time, to determine the effectiveness of aparticular compound the concept of toxicology was developed. Toxicology basically is defined as the study of the effects of chemical agentson biological material with special emphasis on the harmful effects. After gaining 127
    • relevant information on the harmful effects of a compound the levels for its safe usage orthe degree of its safeness is established, this is also known as its (compound) Biosafetylevel. A central concept of toxicology is that effects are dose-dependent; even water canlead to water intoxication when taken in large enough doses, whereas for even a verytoxic substance such as snake venom there is a dose below which there is no detectabletoxic effect.Experimental Review: The materials and methods of the study or the study design in which therationale of the Species selection,animal house facility,age of rats,steps to be followedbefore while and after experiments,group sizes,route of administration,dosageschedule,acute and subacute toxicity methods,measurements and observations to bemade,laboratory parameters, terminal studies and statistical methods are reviewed wideinfra to be updated in the procedural concepts. 128
    • DISCUSSION ON PHARMACEUTICAL METHODS:- “The nature of the chemical bond is the problem at the heart of all chemistry”Discussion on Malla Shodhana: About 50 gm made into small pieces of Malla was tied in a clean, white, squareshaped kora cloth measuring 10 inches in length and 10 inches in breadth.A strongthread was used to tie the pottali and this pottali was suspended in the pot containingKaravellaka swarasa (pH 6) using a wooden rod. Mild heat (Mrudvagni) was given for6hrs (2 yamas). Karavellaka Swarasa was added as and when required to maintain theminimum liquid level. After 6hrs (2 yamas), heating was stopped and the contents wereallowed to self-cooling. Then the pottali was removed from the liquid and washedthoroughly with hot water.After shodhana Malla appears dull white,opaque solid.The above method of shodhana can be rationalized in following ways: Size of the compound particles: The Malla is broken into pieces before placing it for shodhana. As the perimeter of the bigger sizes is in larger units. The acid particles can only collide with the edge of the chip. However, if we break up the large chip into smaller chips. Notice how the acid in the second diagram can reach what used to be the centre of the large chip. Reducing the size of particles increases the rate of a reaction because it increases the surface area available for collisions to take place. This increases the number of collisions. It has no effect on the energy of the particles. Fig 62 : Shows the compact and fragmented particles. Why Pottali? Malla is tied in a Pottali as Arsenic trioxide is highly volatile and because of its O2 content which is paramagnetic, therefore to prevent this reaction with the external environment,it is tied in layers of cloth having minute pores for the swarasa to enter. 129
    • Spatial order: Two boxes of materials separated by a membrane so that the material can diffuse between the 2 boxes. The pottali made of cloth which acts as a membrane is allowed to swing inside the hot juice bath so that the collision between the compound and the solution takes place from all directions. Ion exchange : Ions between a solution and a solid highly insoluble body in contact with it. The solid (ion exchanger) must of course contain ions of its own, for the exchange to proceed sufficiently rapidly, the solid must have an open permeable molecular structure so that ions and solvent molecules can move freely in & out.Malla the solid molecule allows such a process.Karavellaka swarasa acts as a chelating ion exchanger in which the chelating groups are incorporated and are attracted to the Malla matrix as interionic effects are not negligible even for weak acids. Therefore a complex ion is formed by the union of a simple ion with either other ions of opposite charge/neutral molecules.Since Phosphorous is a constituent of Karavellaka swarasa it inhibits the trimethylarsine formation,a toxic metabolite.Infact ‘P’ also helps in arsenic uptake to the target site. Why 6 hours? Law of Mass action:In Chemical kinetics the Speed of the chemical reaction is proportional to quantity of reacting substances in an optimum time. Three major modes of complex series of Arsenic biotransformation in the environment are redox transformation between arsenite and arsenate,the reduction and methylation of Arsenic, ligand exchange,biosynthesis of organoarsenic species which is obtained in an optimum time and temperature. Karavellaka pH 6 : The swarasa is a weak acid and the degree of dissociation of most arsenic species is strongly influenced by the pH.Arsenic is present in the undissociated state in acidic solutions and in solutions not exceeding pH 8.Also a weak Van-der Waals interaction between the unwanted metal impurities is facilitated to eliminate them from the drug Malla to make it pure.The effect of temperature:- When we increase the temperature at which a reaction is taking place, the particles move more quickly. This has two effects: 1) More collisions take place. 2) When a collision occurs, there is more chance that the collision will lead to a reaction, because the amount of energy is more likely to be greater than the 130
    • minimum amount of energy needed (the activation energy).{Maxwell-Boltzmanndistribution of molecular energies}. Fig: 63 Shows the effect of temperature and reaction process.Activation energy is the minimum energy required before a reaction can occur isthe energy for collision reaction. It is involved in breaking some of the originalbonds. This is the cause of agni sannikarsha for the shodhana process.The rule of thumb that the rate of chemical reactions double for every 10 °Ctemperature rise is the reason why agni is increased slowly from mrudu to madyaand then teekshna.As the time and temperature is increased the consistency of boiling swarasachanges to a thin liquid and later a foamy substance appears on the rim of thepot.According to 2nd law of thermodynamics the entropy of the physical orchemical system and its surroundings must increase with time i.e orderly energyof a system like uniform motion must degrade eventually to the random motionparticles in heat bath.This physio-biological treatment makes Malla biologicallycompatible.The effect of concentration: Fig: 66 Shows the effect of concentration on collision.Increasing the concentration of a solution leads to more collisions (greaterfrequency of collisions) so the rate of the reaction goes up. Karavellaka swarasa isadded repeatedly to allow high concentrations resulting in high rate of reactions. 131
    • Autocatalytic reactions where chemical reactions in which at least one of theproducts is also a reactant i.e the product Malla is a reactant and a catalyst. Theproteins , steroids and enzymes in karavellaka swarasa also act as catalysts.It can be considered as an elimination reaction in which a type of organicreaction in which substituents are removed from a molecule in either a one or twostep mechanism.Biosorption: At a maintained pH , a constant raising temperature and the speed ofparticles the organic solution absorbs the metal content from the compound drugknown by the difference in initial concentration and the metal ion remaining insolution.This gives an idea of the absorption of unwanted impurities of Fe,Sb etcmetal ions being absorbed from the compound Malla making it ultrapure.Hightemperatures wouldn’t allow such reactions to take place.Organic encapsulation : The drug molecule Malla is made safe and bioavailablewhen there is an attempt to prepare and characterize a carrier system to minimizethe toxicity of the drug, retention of the drug and enhances a sustained release likeliposomes.Drug designed to target: Malla is made more assimilable through organicreactions to the system and signalling it to the target.Safe ADME :- As inorganic As compounds have rapid oral absorption theorganic innervation ,conversion into methylated form paves a way for safer forms. 132
    • TO DISCUSS ON ANALYTICAL STUDY Analysis of the drug is necessary to know the physico – chemical, macro andmicro properties and to confirm the purity of the drug. According to ancient parametersthe varna of Malla was changed from Pure white to dull white,its crystal glassyappearance was unseen.It shows the reduction in crystallanity..Physicochemical analysis:To ensure, the essential components, which are present withina predetermined range of composition, physicochemical analysis is necessary.Discussion on Ash value: It is a physical method useful in drug standardization. Ashinginvolves an oxidation of the components of the product. It gives a percentage of inorganicconstituents of the sample. It also helps in judging, identification of sample or purity ofthe drug. The two samples of Malla i.e. Ashuddha Malla and Shuddha Malla wereevaluated for ash value and it was found to possess 8.92% and 8.86% respectively whichindicates that Shuddha Malla contains less Ash value and more amount of bio humanavailable particles. Hence it can be said that, the Shodhita Malla is within the standardlimits.Discussion on Acid insoluble ash: The human metabolic process and pharmaco kineticsdepends upon purity and human acceptable forms of drug. When the ash of the twosamples Ashuddha and Shuddha Malla were mixed with Hydrochloric acid, 0.77% and0.48% amount of acid insoluble ash were detected which are not soluble and digestible inhuman GI tract. As Shodhita Malla contains least of silica etc and insoluble ash material,which can also be ascertained that in the present study the Shodhita Malla dwells in thedrug standard tests and hence safe. Hence it can be said that, the Shodhita Malla arewithin the predetermined range.Discussion on water soluble ash: Negligible differences are seen in the determinationof water soluble ash and as it is insignificant in case of inorganic substances, Malla hasshown negligible values before and after shodhana.Discussion on loss on drying at 1100C: The moisture content of any pharmaceuticalagent spoils not only the drug activity but also everything. Loss on drying at 1100C is aphysical test to detect the percentage of moisture content and hence the shelf life of thesample. Lesser the loss on drying at 1100C, the better will be the drug. In the presentstudy, the two samples of Malla i.e. Ashuddha and Shuddha Malla were found to posses 133
    • 0.09% and 0.12% loss on drying at 1100C. Hence it can be stated that it possess leastmoisture content and hence very rare chance of bacterial and fungal growth and also thedrug is having least or nil hydroscopic activity and the drug deterioration chance orcontaminations chances etc. are very less. Concurrently it can be stated that the shelf lifeof the Shodhita Malla in the present study is more and are within the predeterminedrange.Discussion on Loss on ignition:Loss on ignition of Ashuddha Malla was 82.84% andShuddha Malla was 81.39%.Its a known fact that Arsenic is highly volatile andsublimates easily into air. But still this test shows that even at high temperatures ShuddhaMalla is having less amount of loss and Ashuddha Malla shows more amount of loss. Itshows that after shodhana the drug is more stabilized.Discussion on pH: The pH value was estimated for the two samples of Malla i.e.Ashuddha and Shuddha Malla. The pH of the two samples was found to be 6.98 and 7.08respectively. The Ashodhita Malla is within acidic range or it is a weak acid. But theKaravellaka Shodhita Malla revealed alkalinity or can be considered a weak base. Theweak acid and a weak base form a neutral pH which is the need for suitable biologicassimilation.Discussion on Arsenic estimation: The two samples of Malla i.e. Ashuddha andShuddha Malla when subjected to AAS for the estimation shows that it containsqualitatively and quantitatively 69.7% and 74.22% of Arsenic (As%) respectively. Thegenuine drug taken from the market is confirmed for its quality i.e Ashuddha Malla.Thepercentage of Purity is increased in case of Shuddha Malla which is the sign of potentdrug without any other trace metals & impurities that might have adsorbed and detoxifiedby Karavellaka swarasa.Discussion on X- RD:-. According to X- RD patterns composition of Ashuddha Malla i.e. 1st sample isAs2O3 or Arsenolite which is confirmed only after comparing the d-identified peak valueswhich are like fingerprints of the particular sample with d-standard peak values. Thestandard sample which was compared to Ashuddha Malla sample consisted Fe,Mg,Sb,Si0.001 to 0.01% each.Ca and Pb traces and was Isostructural with senarmontite.This givesa clear view of the sample containing impurities which would match to the standards. Composition of 2nd sample i.e. Karavellaka Shodhita Shuddha Malla is As2O3,Arsenolite which is confirmed after comparing d-identified peak values with d-standard 134
    • peak values.The standard which was compared to Shuddha Malla sample consisted of nosuch impurities and was a pure Arsenic trioxide.Analyzing the above two factors we canassume the biological purification method which is undertaken due to Shodhana. Though there is slight shift in the peaks between both the samples it is significantas it shows such a difference.The d-spacing values might be in a second decimalsdifference but it also suggests the change after shodhana with the innervation of mediaand process. The crystal lattice structure of both the samples were Face Centered Cubic butonly the difference was in the unit cell volume between the two.This Change also is asign of stoichiometric differences after shodhana. The count of the Ashuddha malla is about 6858 and that of Shuddha Malla isabout 4771 which signifies the reduced crystallanity of the latter after Shodhanaprocedure. The pharmacological need is the ease of drug ADME, lesser the crystallanitybetter is the absorption.Discussion on Particle Size: The Particle Size was done using Laser Diffractiontechnique which is the latest version of analysis.The particle size of Shuddha Malla afterShodhana has increased which indicates its organic encapsulation to target themicrosphere drug molecule for safe slow and sustained absorption.As particle Size andparticle size distribution can influence the bioavailability,principally by changes insurface area of the drug which is exposed to the acid degradation it is one of essentialfactors. 135
    • Discussion on ACUTE STUDY OBSERVATIONS:- The behavioural profile shows the awareness of the rats initially and later its passivity shows depression. Restlessness shows the mood of the animal and the touch response shows its motor activity. The neurological profile shows central excitation, motor in co-ordination, muscle tone and reflexes. The autonomic profile shows the muscarinic activity which is a sign of sympathetic stimulation. The general sign shows CNS excitation/sympathomimetic action.The reduced food intake is may be due to chronic pain.The retreat to the corner of the cage are examples of distress in anticipation of an experimental procedure. Summarizing the Profile data when evaluated shows the basal score of all other groups to be normal except group IV and VII which shows a abnormal sign which is a sign of absolute toxicity in higher escalated doses. Discussion on Body weight:- Considerable decrease (less than 5%) in body weight of the rats were noted by 3rd day of dose administration in all the test groups except control may be because of the drug Arsenic trioxide (Malla) which acts in detoning the muscular protein mass. Group IV and VII have a significant decrease in body weight which shows that the condition is deteriorating due to escalated doses is a warning of the preceding death. Though group VI shows a suggestive decrease in weight, group III seems to be stable i.e though the dose of Ashuddha Malla & Shuddha Malla are similar this difference is considerable. Significant weight gain in all groups by 12th-14th day is seen except in group VII that cannot be noted because of the death. This may be because Arsenic Trioxide has the threshold effect after certain doses.Discussion on Impending Death :-The rats which were dead in group IVand VII had an impending death as they were in moribund state or death is expectedprior to the next planned time of observation. Signs indicative of this state in rodentsincluded convulsions, recumbency, and tremor. Even the 10,20,30 and 35 times the therapeutic dose of Shuddha Malla shows no toxic sign. The 25 times the therapeutic dose of Ashuddha Malla is toxic. 136
    • The median lethal dose of Shuddha Malla is 16.65mg where as that of Ashuddha Malla is 12.96mg which shows the variable dose significance due to Shodhana. The humane end point was seen at the dose of 14.4 mg of Shuddha Malla whereas at the dose of 10.8 mg in case of Ashuddha Malla.This shows the vitality of the Shodhitha Malla as a Safer drug. The first point along the graph where a response above zero is reached is usually referred to as threshold-dose,therefore the threshold dose of Shuddha Malla is around 12.6mg where as that of Ashuddha Malla is about 9mg.It is also a mark of evidence for the Shoditha Malla to be Safer. The stronger a particular substance is,the steeper the curve will be.Between the two Samples Shuddha & Ashuddha Malla the latter is more steeper.Discussion on Dose-response relationship: Greater the therapeutic ratio, Safer is the drug.The therapeutic index of the drug Shuddha Malla is greater when compared to that of Ashuddha Malla helps us to make the risk-benefit comparisons between the potential drugs. According to some research the no toxic effect dosage obtained in a rodent toxicity may be divided by 10 which is equal to the maximum dosage for clinical use. Therefore the Shuddha Malla safe dose may be 12.6÷10= 1.26mg which falls into the classical dose range. The therapeutic ratio must be atleast more than one for it to be safe. Though both Shuddha & Ashuddha Malla indices are greater than one and proves to be in Safer limits at the prescribed therapeutic drug doses Shuddha Malla is significantly better & safer than Ashuddha Malla and to the degree to which the clinical dose schedule may be varied.Discussion on Blood Parameters:-Haemoglobin (Hb):The marginal decrease in all the groups except in control & that with escalated doseof Shuddha Malla might be because of the degradation of fusion protein due to theinteraction of ATO.The significant decrease is a sign of anaemia which is observed with a single dose ofShuddha & Ashuddha Malla is because haemodilution action of ATO.As a compensating process when Shuddha Malla is administered in repeated doses ithas shown a marginal increase which is a sign of normal regeneration. 137
    • As ATO always has an affinity towards the blood proteins, the concentration of Hb% is increased in a faster rate in escalated doses group when compared to others. Comparing daily therapeutic doses of Ashuddha & Shuddha Malla the Hb% decrease is to lesser extent in the latter. The percentage of increase shows the oxygen carrying capacity being stimulated which is seen in Shuddha & Ashuddha Malla daily therapeutic doses where the increase is much better in the former. Decrease in Hb in the Ashuddha Malla escalated dose reveals the form of aplastic anaemia due bone-marrow depression.Total Leucocyte count : Leucocytosis is observed in Ashuddha Malla escalated group in 10.8mg dose which may be the result of disruption of margination of leucocytes along the vascular endothelium. Leucocytosis is also observed in Shuddha Malla escalated dose of about 23.4mg.Differential Counts : Moderate increase in polymorphs control may be due to stress related to experimental procedures and environmental conditions. An abrupt increase in polymorphs is seen in Ashuddha Malla daily therapeutic dose is a sign of toxicity. Though a negligible decrease is seen in lymphocytes it is a sign of immunosuppression in all the groups due to experimental stress related. A marginal increase by 14th day at 23.4mg (65 times the therapeutic dose) Shuddha Malla group is a sign of lymphocytosis. An eosinophilic condition exists in the Shuddha malla & Ashuddha malla escalated doses wherein which the the condition is better in the former drug. Even in single doses of Ashuddha & Shuddha Malla eosinophilia is seen but the latter being better than former. The daily therapeutic doses of Ashuddha & Shuddha Malla shows a significant difference where it is only a negligible increase in eosinophils in Shuddha Malla daily doses but an absolute eosinophilic condition in daily doses of Ashuddha Malla may be because the recovery process happens in the former but not in latter. Ashuddha Malla at 25 times & Shuddha Malla at 45 times the therapeutic dose group shows Monocytopenic condition in escalated doses may be due to bone marrow failure and aplastic anaemia. 138
    • Packed Cell Volume: The increased haematocrit condition in control may be due to haemoconcentration and decrease in haematocrit in all other groups may be due to hemodilution and expansion of plasma volume. In Shuddha Malla daily therapeutic doses negligible increase is observed by 14th day after decrease by 7th day reveals a recovery or compensation of RBC counts. The decreased RBC count in Shuddha Malla & Ashuddha Malla escalated doses, a single dose and repeated doses of Ashuddha Malla may be due to haemodilution and anaemic condition.Discussion on Serum Parameters :- Increase in Serum urea in both the escalated groups of Ashuddha 35 times & Shuddha Malla 60 times is a sign of acute renal failure,azotaemic condition. Ashuddha Malla at 20 times and Shuddha malla at about 55 times the therapeutic doses shows the moderate increase in creatinine levels which is a sign of impaired of renal function. Ashuddha Malla in escalated doses of 25 times the therapeutic dose has shown increase in SGOT levels which signifies increase in tissue injury to liver as well as to other tissues like in myocardial infarction. A marginal increase in 55 times the therapeutic dose of Shuddha Malla, a single dose and the daily repeated dose of Ashuddha Malla reveals minor enzymatic changes. Ashuddha Malla at 30 times therapeutic dose with highly significant increase in SGPT which is specific for liver tissue due to liver cell injury. The same condition exists in Shuddha malla escalated doses but in the dose of 65 times the therapeutic dose which shows mild enzymatic changes.Discussion on Histopathology :- Normal architecture of all organs are seen in Control group, single dose ofShuddha Malla and Ashuddha Malla except the lung architecture which has a mildcongestion due to ether anesthesia before decapitation or sacrificing. There is a mildlesion in gastric mucosa in the group of Shuddha Malla daily therapeutic dose which maybe due to irritant action of the drug. The escalated doses i.e the 65 times therapeutic doses of Shuddha malla haveshown the toxic markers where there is destruction of cardiac fibres , mild congestion anddegenerating neurons in brain,mild fatty infiltrations of liver with severehaemorrhage,mild congestion and degeneration in kidney tissues,lungs with 139
    • oedema,haemorrhage,infiltraton of inflammatory cells,mild congestion in spleen,gastricmucosa with mild keratinization and descumated krypts. With repeated daily doses of Ashuddha Malla there is congestion of cells inheart,mild lesion and haemorrhage in brain,periportal infiltration of liver,atrophicglomeruli in kidneys,severe haemorrhage around the vessel in lung tissues,destruction ofvilli in gastric mucosa which are the absolute markers of toxicity.The escalated doses i.ethe 30 times therapeutic dose of Ashuddha malla shows a severehaemorrhage,destruction in cardiac fibres,specially with periartirits of lungs,proliferationof lymphoblasts in spleen show the signs of a highly toxic drug than Shuddha Malla inescalation. Discussion on SUB- ACUTE STUDYOBSERVATIONS: The behavioural profile shows the awareness of the rats,hyper activity is due tothe CNS stimulation which is observed in repeated doses of Ashuddha and ShuddhaMalla.The colour change in faecal matter may be due to excessive output of inorganicATO after the required assimilation.Discussion on Body weight:- As a state of drug interaction on muscle tissues where ATO has its affinity towards protein groups the decreased weight is seen in all groups except control by 3rd day. There is a failure to gain body weight in repeated doses of Ashuddha Malla when compared to Shuddha Malla repeated doses which is a sign of an adverse effect. Even with a single dose of the test drug there is a variance between the Ashuddha and Shuddha Malla where the latter is faster in recovering or gaining weight.Discussion on Blood Parameters:- Since the drug ATO has a direct effect on capillary permeability there is a suggestive significant decrease of Hb in groups of single doses of Shuddha & Ashuddha Malla. 140
    • Though the haem-concentration is compensated in faster rate in repeated dose of Shuddha Malla it would take still later days in case of Ashuddha Malla. Leucopenia is observed in repeated doses of Ashuddha malla within 14-21 days of drug schedule which is not seen in Shuddha Malla doses which is a significant change .This may be because of the form of the drug encapsulation made organic and safer for absorption. No comparable changes are observed in DC- polymorphic changes. Repeated doses of Shuddha Malla shows a constant slower rate of decrease in lymphocytes whereas the Ashuddha Malla has an abrupt decrease which is the sign of immunosuppression in a faster rate. A descending type of eosinopenia is seen in Ashuddha Malla single dose where this sign is seen only by 28 days in case of Shuddha Malla. An absolute eosinopenia is seen in Ashuddha Malla daily therapeutic dose by 14th day which is a sign of decrease in ACTH or acute infections which is unseen to this extent in case of Shuddha Malla. The monocytosis stage is observed which suggests of any vascular disease in Ashuddha Malla repeated doses of Ashuddha Malla which shows a linear increase as days pass on but in Shuddha Malla though there is such a stage it compensates within next few weeks. The highly significant decrease in RBC in Ashuddha Malla repeated therapeutic doses is a sign of lesser number of circulating red cells which gives a gross picture of the toxic effects on haemopoiesis or matured circulating cells. The haematocrit value has a negligible change and therefore not of much consideration.Discussion on Serum Parameters:- A highly significant increase of SGOT in a faster rate due to Ashuddha Malla signs an abnormal liver tissue and necrosed cells, also the cardiac tissue injury. There may be mild enzymatic changes in case of single doses of both the drug samples as ATO reacts rapidly with enzymes. Both the single dose and repeated doses of Ashuddha Malla shows a increase in SGPT by 21-28 days is a suggestive sign of liver toxicity. 141
    • The SGPT increase in Shuddha Malla single and repeated doses is less compared to that of Ashuddha Malla.This shows the difference of the drug pharmacology. The decrease in albumin in single dose of Shuddha Malla and Ashuddha Malla shows may be due to haemconcentration. The increase levels in albumin in repeated doses of Shuddha Malla is a sign of maintenance of osmotic pressure needed for proper distribution of body fluids between intravascular compartments and body tissues. Though there are similar levels seen in case of Ashuddha Malla initially there are fluctuations in later days which otherwise is a sign of disturbance in circulation of body fluids. The increase in repeated doses of both Shuddha Malla by 28th day & Ashuddha Malla by 14th day is a sign of glomerular filtration rate though the latter seems to be more nephrotoxic or renal insufficiency. Repeated doses of Ashuddha Malla showing a sign of increase in BUN shows the is a marker of renal function and the state of anti-diuretic hormone.Discussion of Histopathology results:- Repeated dose of Ashuddha Malla showing the oedematous fibres ,severe oedema in heart is a sign of intense toxicity leading to myocardial damage, when compared to that of Shuddha Malla which shows only mild changes in tissue. Congestion of brain tissues and infiltration of few mononuclear cells is a sign of toxicity of repeated doses of Ashuddha Malla in repeated doses which had already started by 14th day. Highly toxic is the state of liver tissues especially to the hepatic parenchyma in repeated doses of Ashuddha Malla where this process has started by 14th day. Even with single dose of Ashuddha Malla a negative sign is seen on 28th day with increased kupffer cells. Mild fatty changes of Liver in repeated doses of Shuddha Malla may be due to specific pharmacology in rats for As. The tubular changes and necrosis in kidney tissues is a sign of absolute toxicity due to Ashuddha Malla repeated doses where severe congestion is already seen by 21st day where these signs are absent in case of Shuddha Malla is a good sign of positive approach of the processed drug. 142
    • Mild congestion of lungs is very common in all groups as seen which is becauseof the procedural effect i.e the ether anaesthesia which is a sign of stress on lungsand the respiratory system.By 28th day mild congestion and haemorrhage of spleen in Ashuddha Malla is thesign of toxicity to haemopoetic system .Repeated doses of Ashuddha Malla shows a toxic profile of gastric mucosa dueto keratinization by 21st day but the positive fact by the same period in case ofShuddha Malla is the regenerative changes or more of basophilic cells seen intissues of gastric mucosa.A sort of replenishing and recovery process is seen atthis stage. In a nut shell as the metabolites of ATO have uncertain biologicaleffects it is still an unraveled fact about its hit target mechanism.The methylationof ATO rapidly to dimethylsarsenic acid and hepatic methylases helps themechanism of metabolic detoxification.Research studies show that an i.p dose of2mg and oral dose of 5 mg results indicate no toxicity. The methyl transferasesinvolved in the biomethylation of As have interesting properties since they have a“hidden” redox component..The administration of the drug Shuddha Malla withspecific anupanas which may act as adjuvants or synergists may take the drugmolecule safely. As rasoushadies classically has been adopted for amandala/ardha mandala as per some experts it is a brilliant way of tackling withthe potential toxic drugs. Shuddha malla maintained as a consolidation therapywould definitely prove its efficacy safely. Though 125mg is mentioned as a lethaldose, when MTD of rat’s dose extrapolated to humans it is about 140mg for thesample taken which shows that our seers were careful enough in indicating eventhe lethal dose. 143
    • CONCLUSIONMalla is a drug with wide therapeutic indications with the least dose mentioned inclassicals and also mentioned cautiously 1 ratti to be the maaraka matra.As a part of purification and Safe drug potentiating mechanism thepharmaceutical procedure is the Swedana using Karavellaka Swarasa.Quantitative analysis of the drugs Ashuddha & Shuddha Malla reveals morepercentage of purity of Arsenic trioxide in the latter.From X-RD study, composition of both the samples Malla was Arsenolite (As2O3)but identified by two different JCPDS standards.The Particle Size of the Shuddha Malla is more than Ashuddha Malla due toorganic innervations after shodhana.The median lethal dose of Shuddha Malla is 16.65mg where as that of AshuddhaMalla is 12.96mg which shows the variable dose significance due to Shodhana.The 5 times the dose(1.8mg) of therapeutic dose (0.36mg) proves Shuddha Mallato be non-toxic in rats.The 10 times the dose (3.6mg) of therapeutic dose proves Shuddha Malla to benon-toxic in rats.The 20 times the dose (7.2mg) of therapeutic dose of Shuddha Malla shows notoxicity.The 30 times the dose (10.8mg) of therapeutic dose of Shuddha Malla shows notoxicity.The 35 times the dose (12.6mg) of therapeutic dose of Shuddha Malla shows notoxicity. 144
    • The 40 times the dose of therapeutic dose i.e. about 14.4 mg of Shuddha Malla isa toxic dose in rats.In the repeated therapeutic doses for 14 days Shuddha Malla is non-toxic whereasAshuddha Malla proves to be toxic.In the 28 days repeated therapeutic dose Ashuddha Malla is highly toxic, thoughShuddha Malla shows some of considerable differences in liver architecture, theregenerative changes proves it to be non-toxic.The therapeutic index of Shuddha Malla is 3.08 which prove Shuddha Malla asnon-toxic.For the sample of Shuddha Malla taken the maximum tolerated dose is 12.6mg forrats.Shuddha Malla is a very safe non-toxic drug in the classically indicated dosagelimits as per acute and Sub-acute repeated dose studies. 145
    • SUMMARYThe present study entitled “Pharmaceutico-Analytical and toxicological study ofShuddha Malla on Wistar albino rats (An experimental study)” - summarized brieflyin Literary, Pharmaceutical, Analytical and Experimental parts.Objectives: 1) Shodhana of Malla by swedana method. 2) Physico – chemical Analysis of 2 samples of Malla i.e. Ashodhita Malla and Karavellaka Shodhita Malla. 3) To find the median lethal dose of Shuddha Malla. 4)To screen the probable toxicity of Shuddha Malla in repeated doses. 5) To screen the probable toxicity of Shuddha Malla in a single dose.Introduction: This part reveals the need for study, aims & Objectives and hypothesis behindselecting Malla for the toxicity study.Literary study: It comprises the literatures on Malla described classically in Rasa texts and alsomodern information of Malla (Arsenic Trioxide). It also includes review on theassociated Dravyas used for shodhana such as Karavellaka.. Pharmaceutical review reveals the concept of Shodhana, Swedana along with theYantras like Dolayantra and Khalva Yantra, used in present study. Analytical review includes Physico – Chemical, Analytical methods like TotalAsh Value, Acid Insoluble Ash,Water insoluble ash, pH, Loss on Drying at 1100C ,Losson ignition ,XRD,AAS and Particle Size. Review of literature also includes the concept of toxicology and the current updated literature regarding its regulatory guidelines. Experimental review reveals the study protocol, Albino rats and its behavior,housing, feeding, environment and also drugs and equipments used in Experimentalstudy.Methodology:Pharmaceutical study: 146
    • It reveals Shodhana of Malla using Karavellaka Swarasa by swedana procedure for 6 hrs.Analytical study: Analysis of 2 samples of Malla i.e. Ashuddha Malla and Karavellaka ShodhitaShuddha Malla . Ashuddha Malla was Pure white and glassy.The Shuddha Malla is dullwhite and opaque. Minimum weight loss occurred after Shodhana. Modern physical tests were conducted on 2 samples of Malla i.e. Ashodhita Mallaand Karavellaka Shodhita Shuddha Malla such as Ash value, Acid Insoluble Ash,Waterinsoluble ash, Loss on Drying at 110 ° C,Loss on ignition and pH. The results were asfollows. Ash values of 2 samples of Malla: Ashuddha Malla – 8.92%, Shuddha Malla –8.86%. Acid Insoluble Ash values of 2 samples of Malla: Ashuddha Malla – 0.77%,Shuddha Malla -0.48%. Water insoluble Ash values of 2 samples of Malla : Ashuddha Malla – 0.04%,Shuddha Malla -0.05%. Loss on Drying at 110°C values of 2 samples of Malla: Ashuddha Malla -0.09%, Shuddha Malla - 0.12%.. pH values of 2 samples of Malla: Ashuddha Malla - 6.98, Shuddha Malla - 7.08. Quantitative analysis for As% of 2 samples of Malla i.e. Ashodhita Malla andShuddha Malla were 69.72% and 74.22% respectively. X-Ray diffraction studies were conducted on 2 samples of Malla i.e. AshodhitaMalla and Shuddha Malla and composition of both the samples was Arsenolite (As2O3),which is confirmed only after comparing d-identified values with d-standard & peakvalues with the face centered cubic crystal lattice. The Particle Size analysis by Laser Diffraction technique shows the size ofAshuddha Malla as 23.67µm and Shuddha Malla as 43.32µm.Experimental study:Acute Study:- The Study period was for 14 days. Seven groups of 8 animals in each group.The groups were Control, single dose of Shuddha Malla, Single dose of Ashuddha Malla,repeated therapeutic dose of Shuddha Malla, repeated therapeutic dose of Ashuddha 147
    • Malla, escalated doses with multiples of 5 for each day for 14 days of Shuddha &Ashuddha Malla to find the median lethal dose. Evaluating the statistics of body weight, haematology & blood Chemistry,viewing histopathology parameters it shows that the escalated doses have proved absolutetoxicity. The median lethal doses of Shuddha Malla and Ashuddha Malla are 16.65mgand 12.96mg of respectively. The 10, 20, 30 and 35 times the dose of therapeutic dose ofShuddha Malla proves to be non-toxic. In the repeated daily therapeutic doses for 14 daysAshuddha Malla has toxic signs which are not found in Shuddha Malla.Sub-acute Study:- The study period was for 28 days. There were 5 groups with 8 animals each.The Control, Single dose of Shuddha Malla, Single dose of Ashuddha Malla, repeatedtherapeutic dose of Shuddha Malla and Ashuddha Malla for 28 days. The body weight, haematology, blood chemistry and histopathology parametersshow that in the dose schedule of 28 days, considerable toxic sign is found only in liverarchitecture in repeated daily therapeutic dose of Shuddha Malla.In repeated therapeuticdaily dose of Ashuddha Malla is proven to be highly toxic accounting all the parameters.The noticing factor is that the Shuddha Malla in repeated daily therapeutic doses showsregenerative changes which are an auto-defense mechanism of the drug and bodyinteraction. In a nut shell Shuddha Malla is a non-toxic safe drug as per drug dosementioned (1mg-4mg) in classicals. 148
    • SCOPE FOR FURTHER STUDYSimilar studies can be done with other Shodhana procedures of Malla.Sub-chronic for 90 days & chronic toxicity tests for 6months should be carriedout to disprove toxicity claims.Comparative study can be done by taking all similar and dissimilar kinds ofShodhana procedures of Malla (swedana, Pachana, Putapaka, etc).Comparative Analytical, Experimental and Clinical study can be tried forShuddha Malla and Malla Bhasma.To analyze the structure of Shuddha Malla, NMR spectroscopy, Massspectroscopy etc can be studied.Comparitive toxicology study of Shuddha Malla & Malla Bhasma prepared byvarious methods can be taken.Secondary Markers, such as levels of insulin or activated T-cell populations canbe used to study the toxicity of Shuddha Malla apart from conventional factors.Phase-I & Phase II Clinical studies of Shuddha Malla can be undertaken.With the collaboration of experimental pharmacology, various experimentalstudies can be undertaken as Anticancer, Immunomodulator etc., to prove itsefficacy. 149
    • LIMITATIONS1. It was a time bound Research work.2. The work was not monetarily sponsored.3. Specific instrumentation and technological accreditation were taken from outside accredited laboratories.4. Specific laboratory assistance for results was taken from pathological labs.5. The rat ADME profile for Arsenic may differ though it is pharmacologically similar to that of humans for the need of extrapolation. Apart from this all the efforts were made to give a prompt justice to the study with thelimited facilities. But the loopholes in the study may be excused by the learned.. 150
    • SUB-ACUTE TOXICITY Fig 55: HEART GROUP I GROUP III 28th Day -Normal 28th Day- Mild Infiltrations. GROUP V GROUP V14th Day Mild Congestion 28th Day Oedematous Fibres GROUP V 28th Day Severe Oedema Fig 56:BRAIN GROUP I GROUP III 151
    • 28th Day Normal 28th Day Mild Congestion GROUP V GROUP V14th Day Congestion of Cells. 21st day Mild Degenerative Changes GROUP V GROUPV28th Day-Mild Congestion 28th Day -Mild Congestion & Infiltration of Few Mononuclear Cells. Fig 57: LIVER GROUP I GROUP III 152
    • 28th Day -Normal 28th Day - Fatty Changes Degeneration Of Hepatocytes. GROUP IV GROUP V28th Day- Increased Kupffer Congestion & HaemorrhageCells. (Modulatory Effect) 14th Day GROUP V GROUP V21st Day-Fatty Changes 28th Day-Severe HaemorrhageMild Haemorrhage Necrosed Hepatocytes.Swollen Hepatocytes. 153
    • GROUP V GROUP V 28th Day-Hepatocytes 28th Day-Hepatocyte Degeneration Separated,Congestion. Fig 58: KIDNEY GROUP I GROUP III 28th Day - Normal 28th Day-Mild Congestion GROUP V GROUP V21st Day-Severe Congestion 28th Day-Necrosis,Tubular Changes. Fig 59: LUNG GROUP I GROUP V 154
    • Mild Congestion & Oedema 28th Day-Mild Emphysema Fig 60: SPLEEN GROUP I GROUP V 28th Day-Normal Mild Congestion & Haemorrhage 28th Day Fig 61: GASTRIC MUCOSA GROUP V GROUP IIIKeratinization with. Regenerative Changes MoreSlightly Proliferting Cells with More Basophilic Cells. 155
    • ACUTE TOXICITY Fig 48: HEART GROUP I GROUP IV Normal Destruction of Cardiac Fibres GROUP VI GROUP VIICongestion of Cells Severe Haemorrhage And destruction of Muscle Fibers. Fig : 49 BRAIN GROUP I GROUP IV Normal 12th Day Mild Congestion GROUP IV GROUP VI 156
    • 14th Day- Mild Lesion Swollen Mild Lesion with Haemorrhage & Degenerating Neurons. 14th Day GROUP VII Congestion, Mild Swollen Oligodendrites. GROUP VI GROUP VI ICongestion, Infiltration of Haemorrhage & InfiltrationLymphocytes, Haemorrhage of Inflammatory Cells. Fig 50: LIVER GROUP I GROUP IV 157
    • 14th Day- Normal Mild Fatty Infiltrations 12th Day GROUP VI GROUP VICongestion of Cells-14th Day 14th Day Periportal Infiltration of Inflammatory Cells. GROUP IV GROUP VIISevere Haemorrhage Hepatocytes Separated Forwith Swollen Hepatocytes accumulation Of Fluid GROUP VII GROUP VII 158
    • Destruction of Hepatocytes Destruction Of Hepatocytes.Remnants of Epithelium. Fig 51: KIDNEY GROUP I GROUP IV Normal Mild Congestion & Degeneration. GROUP VI GROUP VII Atrophic Glomeruli Congestion, Haemorrhage Tubular Changes Necrosis of Tubular Epithelium Degeneration, Necrosis. with Proteinacious Casts. GROUP VII GROUP VII 159
    • Atrophic Glomeruli Swollen TubulesHaemorrhage Atrophic Glomeruli. Fig 52: LUNGS GROUP I GROUP IVMild Congestion & Oedema Oedema,Haemorrhage Infiltration of Inflammatory Cells In Interstitia. GROUP VI GROUP VISevere Haemorrhage Thickening Of AlveolarAround The Vessel wall,Haemorrhage. GROUP VII Periartiritis, Thickening of Alveolar Wall,Haemorrhage & Infiltration of Inflamatory Cells. Fig 53: SPLEEN 160
    • GROUP I GROUP IV. Normal Mild Congestion GROUP VI I GROUP VIICongestion & Haemorrhage Proliferation of Lymphoblasts around The Arteriole. Fig 54: GASTRIC MUCOSA GROUP I GROUP III Normal Mild Lesion GROUP IV GROUP VI 161
    • Mild Keratinization Villi Showing DescumationDescumation of Krypts & Destructionor Gastric Glands. GROUP VII Descumated Epithelium Due To Irritation. PHARMACEUTICAL 162
    • Fig 3: Malla before Shodhana Fig 4: Karavellaka Fig 5: Karavellaka kalkaFig 6: Extraction of Fig 7: Karavellaka swarasa Fig 8: Malla in PottaliKaravellaka juiceFig 9: Dola yantra Swedana Fig 10: Boiling swarasa. Fig 11: After 5 hrs with lather after 2 hrsFig 12 : Pottali opened. Fig 13: Washing Malla pieces. Fig 14: Powdering Malla inafter cooling Khalva. EXPERIMENTAL 163
    • Fig 15:Active rats in cage. Fig 16: Cages arranged. Fig 17: Feeding and water.Fig:18 Dosage prepared. Fig 19: Weighing animals. Fig 20: Analytical balance.Weighing the drug. Fig 21: Numbering the Fig 22: Tuberculin syringe rats.Fig 23: Gavaging dose. Fig 24: Ether Chamber. Fig 25: Storage Vials. 164
    • Fig 26: Prepn of ether Fig 27: Blood drawing from chamber. retro-orbital plexus Fig 28: Capillary tube. Fig 28: Blood in storage vial. Fig 29: Centrifuging machine. Fig 30:Semi-auto analyzer. Fig 31: Dissection set. Fig 32: Diethyl Ether. Fig 33: Micropipettes with SerumFig 34: Clear Serum Fig 35: Reagent mixing. Fig 36: Analyzing serum .Fig 36 Rehabilitating rat after. Fig 37: Blood clot after Fig 38: Separatinganesthesia centrifuge Serum. Dissection of the rats after Sacrificing. 165
    • Fig 39:Rat in ether. Fig 40: Anaesthetized Rat. Fig 41: Decapitation of Rat.chamberFig 42: Dead Rat. Fig 43: Exposing Fascia. Fig 44: Excising the organs.Fig 45: Dissection of Fig 46: Fixing organs in NBF. Fig 47: Ready Brain. Containers 166
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    • 27. Vaidya Sri Lakshmipathi Shasthri, Yogaratnakara, 6th Edition, Edited by Bhishagratna Sri Brahmashankara Shasthri, , Chaukambha Sanskrit Sansthan Publishers, Varanasi, 1997, Vishavarga, 166th pp.28. Sushruta, Sushruta Samhita, Translated by Kaviraja,, Dr. Ambikadatta shasthri, 14th Edition, Chaukambha Samskruta Sansthan, Varanasi, , 2002, Kalpasthana, 2nd chapter, 5th verses, 17th pp.29. Sadananda Sharma, Rasatarangini, 11th Edition, Edited by Kashinath Shasthri, Motilal Banarasidas, Delhi, 2004, 11th chapter, 136th verses, 265th pp.30. Ibid. 11th chapter, 133rd -134th verses, 266th pp.31. Ibid. 11th chapter, 135th verses, 266th pp.32. Ibid. 11th chapter, 137th verses, 266th pp.33. Ibid. 11th chapter, 137th verses, 266th pp.34. Vaidya Sri Lakshmipathi Shasthri, Yogaratnakara, 6th Edition, Edited by Bhishagratn,Sri Brahmashankara Shasthri, Chaukambha Sanskrit Sansthan Publishers, Varanasi, 1997, Vishavarga, 166 pp.35. Ibid. Vishavarga, 166th pp.36. Ibid. Vishavarga, 166th pp.37. Yadvji Trikamji, Rasamritam, Translated by Sri Damodar Joshi, 1st Edition, Varanasi, Chaukambha Samskruta Prakashana, 1998, 14th chapter, 1st verses, 3rd pp.38. Rasavagbhata, Rasa Ratna Samucchaya, With ‘Vijnaanabodhini’ Hindi trans. and commentary by Prof. Dattatreya Anant Kulkarni, Meharchand Lachhmandas Publications, New Delhi, Chaukambha Amara Bharathi prakashana, 3rd chapter, 125th verses, 65th pp.39. Rasachandamshu, Translated in Kannada by Dr. A. Halseekar 1850, Atreya sadana, Ganesh pet, Hubli, Poorvakhanda, 277th verses, 70th pp.40. Ibid. 278th Verses, 70th pp.41. Ibid. 279th - 280th Verses, 70th pp.42. Ibid. 281st Verses, 70th pp.43. Ibid. 282nd - 283rd Verses, 70th pp.44. Bhudeb Mookerjee, Rasajalanidhi, 3rd Edition,Chaukambha Publishers, , 1998, 2nd vol, 3rd chapter, 213th pp.45. Sadananda Sharma, Rasatarangini, 11th Edition Edited by Kashinath Shasthri, Motilal Banarasidas, Delhi, 2004, 11th chapter, 171st-176th verses, 265 pp.46. Rasachandamshu, Translated in Kannada by Dr. A. Halseekar, 1850, Atreya sadana, Ganesh pet, Hubli, Poorvakhanda, 284th - 286th version, 70th pp. 169
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